Cardiomyocyte production

ABSTRACT

Methods and composition for the production of cardiomyocytes from differentiation of pluripotent stem cells are provided. For example, in certain aspects methods including differentiating pluripotent stem cells in a large volume of suspension culture in the presence of ROCK inhibitors are described. In further aspects, methods for differentiation of stem cells into cardiomyocytes that overcome variability between different stem cell clones and different batch of culture medium are provided.

The present application is a divisional of U.S. application Ser. No.14/665,616, filed Mar. 23, 2015, which is a continuation of U.S.application Ser. No. 13/794,679, filed Mar. 11, 2013, now abandoned,which is a divisional of U.S. application Ser. No. 12/907,714, filedOct. 19, 2010, now U.S. Pat. No. 8,415,155, issued Apr. 9, 2013, whichclaims the priority benefit of U.S. Provisional Application No.61/252,919, filed Oct. 19, 2009. The subject matter of this applicationalso relates to that of U.S. Provisional Application No. 61/394,589filed on Oct. 19, 2010. The entire text of each of the above referenceddisclosures is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of stem celldevelopment. More particularly, it concerns the induction ofcardiomyocyte differentiation from pluripotent stem cells.

2. Description of Related Art

Cardiomyocytes are thought to be terminally differentiated. Although asmall percentage of cardiomyocytes may have proliferative capacity, thisis not sufficient to adequately replace injured or dead cardiomyocytes.Death of cardiomyocytes occurs, for example, when a coronary vessel isoccluded by a thrombus and the surrounding cardiomyocytes cannot besupplied with necessary energy sources from other coronary vessels. Lossof functional cardiomyocytes may lead to chronic heart failure.

The proliferative capacity of the cardiomyocytes is not sufficient toregenerate the heart following myocardial injury. Conventionalpharmacological therapy for patients with different stages of ischemicheart disease improves cardiac function, survival and quality of life.However, ischemic heart disease is still the most life-threateningdisease in the United States and Europe; accordingly, alternativetherapies will be necessary to improve the clinical outcome for patientswith ischemic heart disease further. In recent years, the focus on cellreplacement therapy has been intensified, stimulated by the increasingnumber of potential cell sources for transplantation, such as skeletalmyoblasts, adult cardiac stem cells, bone marrow stem cells andembryonic stem cells.

A potential route for restoring “normal” heart function is replacementof injured or dead cardiomyocytes by new functional cardiomyocytes.Pluripotent stem cells, such as human embryonic stem (ES) cells orinduced pluripotent stem cells (iPS) cells, are a potential source ofcells for cardiomyocyte replacement. Differentiation of pluripotent stemcells into cardiomyocytes can be achieved either spontaneously or uponinduction,.

However, a number of obstacles have stood in the way of developing aparadigm for obtaining substantially enriched populations ofcardiomyocyte lineage cells from pluripotent stem cells. Some ensue fromthe relative fragility of pluripotent cells of primate origin, thedifficulty in culturing them, their exquisite sensitivity and dependenceon various factors present in the culture environment, and lowefficiency and wide variation of differentiation methods. Thus, there isa need to improve induction of differentiation of pluripotent stem cellsto cardiomyocytes, especially for large-scale production.

SUMMARY OF THE INVENTION

The present embodiments overcome a major deficiency in the art byproviding methods for differentiating pluripotent stem cells intocardiomyocytes, especially for large scale and high efficiencyproduction to meet the needs in clinical applications.

Procedures for differentiating pluripotent stem cells preferably employculture conditions that attempt to mimic the in vivo environment drivingthe development of a particular lineage, such as by the addition ofspecific growth factors. When differentiated in vitro, a number ofsources contribute to the growth factor environment, including: 1)endogenous expression from the cells themselves, 2) the sera and/ormedia (non-limiting examples include TeSR, mTeSR, RPMI medium,supplemented DMEM-F12 or dilutions thereof) that the pluripotent stemcells are cultured and/or subsequently differentiated in, and 3) theaddition of exogenous growth factors. In regard to the endogenousexpression of growth factors, the inventors have recognized aclone-to-clone variability as well as variability due to differences inthe primary culture of the cells. Furthermore, the inventors havedetermined that there exists (sometimes dramatic) batch-to-batchvariability in the growth factor content of the culture medium used inculturing and differentiation of pluripotent stem cells, bothintroducing unpredictability to cell lineage differentiation.

In order to address the foregoing variability in pluripotent stem celldifferentiation procedures, the present inventors have developed atechnique which accounts for all the sources contributing to the growthfactor environment in a given differentiation culture, which areindependently “balanced” for each batch or lot of medium and for eachindividual stem cell clone that is employed, in order to achieve anoptimal differentiation. This may be achieved by the addition ofdifferentiation factors to modulate developmental signaling pathways.Examples include: 1) the addition of antagonists to reduce the totalsignal in certain pathways, 2) the addition of agonists to increase thetotal signal of certain pathways or 3) combinations of agonists and orantagonists to optimize the signal.

For increased production of cardiomyocytes, there is provided a methodcomprising incubating pluripotent stem cells from a selected cell linein a suspension culture under conditions to promote aggregate formation;and differentiating the stem cells into cardiomyocytes in a cardiacdifferentiation medium prepared from a selected batch of culture medium.For example, the culture medium is TeSR, mTeSR or RPMI mediumsupplemented DMEM-F12 or dilutions thereof. In the case of cardiomyocytedifferentiation, and without wishing to be bound by theory, it iscontemplated that TGFβ signaling pathways may be delicately regulated byadjusting the external addition of certain growth factors to achieveoptimal cardiac differentiation condition. In particular, BMP signalingand Activin signaling are two exemplary TGFβ signaling pathways that canbe optimized for the particular batch of medium and pluripotent cellclone (e.g., from an iPS line) employed. The relative activity ratiobetween the two signaling pathways may be important for the optimalcardiomyocyte differentiation condition. Thus, differentiation factorsmay include positive modulators or inhibitors of BMP signaling and/orActivin signaling. For example, BMP signaling inhibitor comprisesdorsomorphin and Activin signaling inhibitor comprises SB431542.

Thus, in accordance with some aspects of the present invention, thecardiac differentiation medium is prepared from the culture medium byadjusting the level of one or more differentiation factors in theculture medium at amounts determined to be appropriate for cardiacdifferentiation of the selected cell clone or line and culture mediabatch employed. In particular aspects, the selected cell clone is aninduced pluripotent stem (iPS) cell clone. Described herein below(Example 8) is an exemplary procedure for separately determining theappropriate amount of growth factors that should be added for any givenmedium batch and pluripotent cell clone employed. Once the adjustment isdetermined for the particular medium batch and cell clone, theadjustment is incorporated into the differentiation procedure, providinga highly reproducible differentiation procedure tailored for each batchand clone.

The selected cell clone is typically derived from a single pluripotentstem cell in an adherent culture or a suspension culture. For example,it is preferred that the selected cell clone be derived from a singlepluripotent stem cell by a process comprising incubating the singlepluripotent stem cell in medium comprising, for example, aRho-associated kinase (ROCK) inhibitor or a myosin II inhibitor underconditions to promote cell growth. Such a myosin II inhibitor may beblebbistatin. The starting number of pluripotent stem cells may beabout, at least or at most 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ cells or anyrange derivable therein.

In certain aspects, the conditions to promote aggregate formationcomprises externally added ROCK inhibitor, myosin II inhibitor,fibroblast growth factor (FGF) or hepatic growth factor (HGF). Theaggregate formed from the pluripotent stem cells may be about, at leastor at most 5, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400,450, 500 μm in diameter. The diameter may be a mean, median or averagediameter. The suspension culture may have a volume of about, at least orat most 2 ml, 5 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 100 ml, 200 ml,500 ml, 1 liters, 3 liters, 5 liters, 10 liters, 20 liters, 25 liters,30, liters, 40 liters, 50 liters, or any range derivable therein, suchas in a bioreactor.

The cardiac differentiation medium may also comprise externally adjustedfibroblast growth factor (FGF), hepatocyte growth factor or anydifferentiation factors that to be used or screened. Such FGF, HGF, orother differentiation factor like TGFβ signaling modulators may be at anamount of at least, about or at most 0.1, 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200 ng/ml or any rangederivable therein.

For selection or enrichment of desired cells, the pluripotent stem cellsand/or cardiomyocytes differentiated therefrom may contain one or moretransgenes. For example, the one or more transgenes encode a selectableand/or screenable marker under the control of a cardiomyocyte-specificpromoter. The method may further comprise enriching or purifying thedifferentiated cardiomyocytes.

In certain aspects, the differentiation factors whose level is adjustedin the culture medium comprise one or more of modulators of signalingpathways of bone morphogenetic protein, ActivinA/Nodal, vascularendothelial growth factor (VEGF), dickkopf homolog 1 (DKK1), basicfibroblast growth factor (bFGF), insulin growth factor (IGF), and/orepidermal growth factor (EGF). For example, the differentiation factorsmay comprise BMP2, BMP4, BMP10, Activin A, bFGF, IGF, EGF, BMP signalinginhibitor, Activin signaling inhibitor, or a combination thereof. Inparticular, the BMP signaling inhibitor may comprise dorsomorphin; theActivin A signaling inhibitor may comprise SB431542.

In certain aspects, the differentiation medium may have been preparedfrom the selected batch of culture medium by adjusting the amounts ofaddition of one or more differentiation factors, such as fordifferentiation of cardiomyocytes. In further aspects, thedifferentiation medium may have been prepared from the selected batch ofculture medium by adjusting the timing of addition of one or moredifferentiation factors, such as for differentiation of cardiomyocytesas well.

There may provided a method involving separately determining theappropriate amount and/or timing of growth factors that should be addedfor any given medium batch and pluripotent cell clone employed. Once theadjustment is determined for the particular medium batch and cell clone,the adjustment is incorporated into the differentiation procedure,providing a highly reproducible differentiation procedure tailored foreach batch and clone.

In certain aspects, the method may comprise determining amounts ofaddition of one or more differentiation factors appropriate fordifferentiation into selected lineages, such as cardiomyocytes. Thisdetermination may comprise testing differentiation of cells from theselected clone in a culture medium from the selected batch added withvaried amounts of differentiation factors during a test period. Forexample, varied amounts of differentiation factors may be added during atest period. The test period may be the same or varied for a testcondition with a specific concentration of differentiation factors. Forexample, the test period may start from about 1, 2, 3, 4, 5, 6, 7 priorto differentiation or day 1, 2, 3, 4, 5 after differentiation, and endon day 6, 7, 8, 9, 10, 11, 12, or 13 after differentiation (anyintermediate time period may also be included). The same concentrationof differentiation factors may be changed daily or every 2, 4, 8, 16,24, 48 hours or any intermediate intervals.

Such varied amounts of differentiation factors may include conditionswith varied ratios of BMP/Activin signaling activity. This may beachieved by varying amounts of BMP, Activin, BMP signaling inhibitor,and/or Activin signaling inhibitor. For example, this variation mayinclude one or more of the following varying conditions: variedconcentrations of BMP4 alone, varied concentration of Activin A alone,varied concentration of BMP signaling inhibitor, varied concentration ofActivin signaling inhibitor, and varied concentration of combination ofBMP4 and Activin A.

To determine the appropriate cardiomyocyte differentiation condition,the testing may further comprise measuring mesoderm or cardiomyocytedifferentiation efficiency for each condition and selecting thecondition with the highest differentiation efficiency as beingappropriate for the differentiation of the selected pluripotent stemcell clone into cardiomyocytes and selected batch of culture mediaemployed. The measurement of differentiation efficiency may comprisemeasuring mesoderm marker expression at least or about days 5, 6, 7, 8,9, 10, 11, 12, 13 after differentiation or any intermediate time range.Non-limiting examples of mesoderm markers include KDR, PDGFR-a, CXCR4,CKIT^(negative), N-Cadherin, and/or MESP1. In other aspects, themeasurement of differentiation efficiency may comprise measuringdifferentiation efficiency comprises measuring cardiomyocyte markerexpression at about or at least day 14 after differentiation. Afterselection of the appropriate condition, the method may comprisedifferentiating the stem cells into cardiomyocytes in a differentiationmedium prepared from a selected batch of culture medium, wherein thedifferentiation medium has been prepared from the selected batch ofculture medium under the selected condition during the test period.

There may be also provided an isolated cell population of at least orabout 10⁶, 10⁷, 10⁸, 5×10⁸, 10⁹, 10¹⁰ cells (or any range derivabletherein) comprising at least or about 90% (for example, at least orabout 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivabletherein) cardiomyocytes.

In a certain embodiment, there may be provided methods for increasedproduction of cardiomyocytes comprising: a) incubating pluripotent stemcells in a suspension culture under conditions to promote aggregateformation, wherein the suspension culture comprises aggregate promotionmedium; and b) then differentiating the stem cells into cardiomyocytesin a suspension culture suitable for cardiac differentiation. Forexample, the suspension culture suitable for cardiac differentiation mayhave a volume greater than 5 milliliters. The aggregate promotion mediummay contain a Rho-associated (ROCK) inhibitor, In certain aspects, aROCK inhibitor may be a ROCK-specific inhibitor.

The term “increased production” may refer to an increase in the absolutenumber of cells or it may refer to an increase in the percentage ofcardiomyocytes compared to non-cardiomyocyte cells, or both, unlessotherwise indicated. It is contemplated that methods may concern anincrease in the absolute number of cardiomyocytes in some embodiments.In other embodiments, methods may concern an increase in the percentageof cardiomyocytes. In further embodiments, there may be an increase inboth absolute number and percentage of cardiomyocytes.

In a further aspect, the method further comprises: c) pooling multiplesuspension cultures after aggregate formation and/or afterdifferentiation; and d) enriching for cardiomyocytes.

In some embodiments, methods result in a cell population of at least orabout 10⁷, 10⁸, 10⁹, or up to about 10¹⁰ cells (or any range derivabletherein) comprising at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% cardiomyocytes, or any percentage derivable therein.

In another embodiment, there are methods for preparing cardiomyocytescomprising: a) obtaining a pluripotent stem cell population comprisingpluripotent stem cell aggregates; and b) incubating the pluripotent stemcells in a suspension culture comprising cardiac induction medium (alsocalled CIM) to differentiate the stem cells into cardiomyocytes. Thecardiac induction medium may include one or more cardiac inductionagents effective to differentiate the stem cells into cardiomyocytes,such as fibroblast growth factor (FGF). The methods are particularlysuited for large scale production of cardiomyocytes. Pluripotent stemcell aggregates may be formed in a suspension culture, which may bebetween about 0.5 ml and about 25 liters, such as in a bioreactor. Someembodiments involve cells growing in a space whose volume is larger thana standard petri dish or 96-well plate; consequently, some embodimentsexclude the use of such containers.

In certain embodiments of the invention, large scale production ofcardiomyocytes may be implemented. “Large scale,” as used herein, refersto the use of a cell culture of a volume of at least 500 ml with aconcentration of at least 1.0×10⁶ cells/ml. The volume may be at leastor about 500 ml, 600 ml, 700 ml, 800 ml, 1 liter, 2 liters, 3 liters, 5liters, 10 liters, 20 liters, or up to 25 liters, or any range derivabletherein, such as in a bioreactor. Cell concentration in suspensionculture may be at least or about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ cells/ml, orany range derivable therein. Cells may be manipulated subsequent toproduction, such as by concentrating them and/or reducing the cellculture volume.

In certain aspects, for large scale production of cardiomyocytes, thesuspension culture may be moved at a speed of at least or about 10, 15,20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200,250, 300 rpm, or any range of speed derivable therein. The movement maycomprise stirring, shaking, or rotating as non-limiting examples.

The pluripotent stem cells may comprise one or more transgenes; forexample, any steps of the methods may be performed under conditions toselect for transgenic cells. In a further aspect, the cardiomyocytes maybe also incubated in a cardiac maintenance media that lacks added FGF.Steps of the invention, such as step b) described above, may last for atleast 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 weeks, or up to 106 days, or any time range derivable therein. Ina further aspect, the method further comprises: c) pooling multiplesuspension cultures after aggregate formation and/or afterdifferentiation; and d) enriching for cardiomyocytes. In someembodiments, methods result in a cell population of at least or about10⁷, 10⁸, 10⁹, or up to about 10¹⁰ cells (or any range derivabletherein) comprising cardiomyocytes of at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% (or any range derivable therein).

Other embodiments concern methods for preparing cardiomyocytescomprising: a) growing a population of transgenic iPS cells in asuspension culture of more than 5 milliliters comprising aggregatepromotion medium that includes a ROCK inhibitor and FGF under conditionsthat promote aggregate formation; and b) then differentiating the iPScells into cardiomyocytes in a suspension culture comprising cardiacinduction medium that includes FGF under conditions to promotecardiomyocyte differentiation. In certain aspects, the population oftransgenic iPS cells may be clonally derived from a single transgeniciPS cell. In a further aspect, a cell population of at least or about10⁷, 10⁸, 10⁹, or up to about 10¹⁰ cells (or any range derivabletherein) comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% (or any range derivable therein) cardiomyocytes produced by themethod described above, wherein the method further comprises: c) poolingmultiple suspension cultures after aggregate formation and/or afterdifferentiation; and d) enriching for cardiomyocytes.

In certain further embodiments, methods involve pluripotent stem cellsas starting material for cardiomyocyte production or preparation, whichcould be embryonic stem (ES) cells, induced pluripotent stem cells, orembryonic stem cells derived by somatic cell nuclear transfer. In acertain aspect, the pluripotent stem cells may be clonally derived froma single pluripotent stem cell, may comprise a substantial portion ofcells clonally derived from a single cell, or may be a pool of multiplepopulations of cells, wherein each population of cells is clonallyderived from a single cell. An exemplary process for obtainingpluripotent stem cells from a single cell may comprise incubating asingle pluripotent stem cell in medium comprising a ROCK inhibitor underconditions to promote cell growth, such as being incubated underadherent culture conditions. Prior to growing the pluripotent stem cellsin the suspension culture for aggregate formation in the step a), thesingle pluripotent stem cell as the originating source may be passagedonce, twice, three times, four times, or preferably at least five times.In another aspect, the pluripotent stem cells may also be derived froman iPS cell population comprising more than a single cell.

In still further aspects, about 10⁷ to about 10¹⁰ of the pluripotentstem cells may be first incubated in the suspension culture in step a)for aggregate formation. The pluripotent stem cell aggregates may beformed by incubating pluripotent stem cells with aggregate promotionmedium comprising a ROCK inhibitor, which may be about 0.05 to about 5μM, for example, at least or about 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2,2.5, 5 μM, or any concentration effective for promoting cell growth orsurvival, including any range derivable therein.

In certain aspects, a culture medium, such as the aggregate promotionmedium, may comprise fibroblast growth factor (FGF), for example at aconcentration of about 5 to 200 ng/ml, such as at least or about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,150, 180, 200 ng/ml, or any range derivable therein. Optionally hepaticgrowth factor (HGF) may also be included, for example at a concentrationof at least or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 180, 200 ng/ml, or any range derivable therein.

The aggregate promotion medium may further comprise an antibiotic, suchas zeocin, which may be used for cardiomyocyte enrichment or selection.The aggregates formed by pluripotent stem cells prior to differentiationmay be at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400 μm (or any range derivable therein) in diameter;in another aspect, at least about 20%, 30%, 40%, 50%, 80%, 90%, 95%, or99% (or any range derivable therein) of the aggregates may comprise atleast or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 80, 100, 150, 200,250, 300, 400, 500, 1000 cells, or any range derivable therein. Incertain aspects, a substantial portion (e.g., about more than 50%, 80%,90%, 95%, 99% or any range derivable therein) of the aggregates areabout 80 to 200 μm in diameter. The approximately uniformity of anoptimal range of aggregate size may help cardiomyocyte differentiationas differentiation is guided by spatial cues and interaction betweenvarious cell types, which can be manipulated by varying aggregate size.

As in the step of cardiac differentiation, the suspension culture fordifferentiating the stem cells may comprise any cardiac induction mediumsuitable for cardiac differentiation, for example, a cardiac inductionmedium having FGF, which may have a concentration of about 5 to about200 ng/ml, such as at least or about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200 ng/ml or anyrange derivable therein. In a further aspect, the suspension culture fordifferentiating or the cardiac induction medium may not include addedROCK inhibitor.

In a certain aspect, such a suspension culture in step a) or b) of anymethod described above may be at least or about 2 ml, 5 ml, 10 ml, 20ml, 30 ml, 40 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 liters, 3 liters, 5liters, 10 liters, 20 liters, or up to 25 liters, or any range derivabletherein, such as in a bioreactor. Embodiments of the method may furthercomprise pooling multiple suspension cultures, for example, after stepa). For example, multiple suspension cultures of aggregated cells may bepooled prior to differentiating the stem cells, or multiple suspensioncultures of differentiated stem cells, such as cardiomyocytes, may bepooled.

In some further aspects, the pluripotent stem cells, such as transgeniciPS cells, may contain one or more transgenes, such as transgenesencoding a selectable marker, which for example confers antibioticresistance, or a screenable marker, which may be fluorescent orluminescent. The aggregate promotion medium or cardiac induction mediummay comprise an antibiotic that allow for selection of cells expressingthe transgene. For cardiomyocyte selection or enrichment, the transgenemay be under the control of a tissue-specific promoter, wherein thetissue specificity is for cardiomyocytes.

Furthermore, in certain aspects, methods may further comprise enrichingor purifying the differentiated cardiomyocytes. In a certain embodiment,the cardiomyocytes may express one or more selectable or screenabletransgene, wherein the transgene may be used for enrichment orpurification of the cardiomyocytes. For example, the transgene may be anantibiotic resistance gene. For cardiomyocyte enrichment or isolation,such a transgene may be under the control of a promoter that is specificfor cardiomyocytes, such as a myosin promoter or troponin T promoter.

Further embodiments provide an isolated cell population of at least orabout 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ cells (or any range derivable therein)comprising at least 90% (for example, at least or about 90%, 95%, 96%,97%, 98%, 99%, 99.5%, or any range derivable therein) transgeniccardiomyocytes. In a specific example, the cell population may contain atransgene under a promoter specific for cardiomyocytes.

In some embodiment, there may be a cell population of at least or about10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ cells (or any range derivable therein)comprising at least 90% (for example, at least or about 90%, 95%, 96%,97%, 98%, 99%, 99.5%, or any range derivable therein) cardiomyocytesproduced by a method comprising: a) obtaining a cell population from atransgenic induced pluripotent stem (iPS) cell; b) growing thepopulation of transgenic iPS cells in at least one suspension culture of5 milliliters to 25 liters comprising an aggregate formation mediumhaving a ROCK inhibitor and FGF under conditions that promote aggregateformation; c) optionally pooling multiple suspension cultures containingiPS cell aggregates; d) then differentiating the pooled iPS cellaggregates into cardiomyocytes in a suspension culture comprisingcardiac induction medium having FGF; and, e) enriching forcardiomyocytes.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: NaCitrate Cell Splitting/Double Stack Scale up experimentaldesign.

FIGS. 2A-C: Double times for iPS 6.1 and iPS 6.1 MRB. These graphs showthe doubling time of Dispase-split T150 control cells as well asNaCitrate split Double stack cells for both iPS 6.1 and iPS 6.1 MRB.

FIG. 3: Karyotype testing results performed on both iPS6.1 and iPS6.1MRB's that were maintained in Double Stacks split with NaCitrate. FIGS.1A-B: Marking of ULA T75 or T25 flasks.

FIGS. 4A-D: Aggregates (Day 14) formed from IPS6.1 cells in DoubleStacks (DS) and T150 flask.

FIGS. 5A-C: Aggregates (Day 14) formed from IPS6.1 MRB cells in DoubleStacks (DS).

FIGS. 6A-B: Aggregates (Day 14) formed from IPS6.1 MRB cells in T150flask.

FIGS. 7A-C: 2nd round iPS MRB Day 14 Aggregates from DS.

FIGS. 8A-C: Flow cytometry pluripotency analysis of iPS6.1 and iPS6.1MRB cells after four or seven passages with both dispase and NaCitratecell splitting.

FIG. 9: Compiled data of cardiomyocyte cells generated from iPS6.1 andiPS6.1 MRB cells passaged with dispase in T150 flasks (control) and inDouble stacks with NaCitrate.

FIGS. 10A-B: Illustration of marking of feeding line in flasks.

FIGS. 11A-B: Illustration of aspiration of media from flasks.

FIG. 12: IPS single cell density experimental design.

FIGS. 13A-B: Count of initial iPS cells incorporated into aggregates.

FIG. 14A-E: Flow cytometry analysis of differentiated cardiomyocytes bycTNT staining.

FIGS. 15A-D: Count of cardiac cells differentiated from differentinitial iPS cell densities.

FIGS. 16A-C: Images of iPS cells in rotary culture.

FIGS. 17A-D: Images of cell aggregates.

FIG. 18: Experimental Design 1 of single cell aggregate formation forcardiomyocyte formation in H₁₁₅₂.

FIG. 19: Experimental Design 2 of aggregate formation for cardiomyocyteformation using cells maintained with dispase.

FIG. 20: Aggregate formation images from Experimental Design 1 of singlecell aggregate formation for cardiomyocyte formation in H₁₁₅₂.

FIG. 21: Aggregate formation images from Experimental Design 2 ofaggregate formation for cardiomyocyte formation using cells maintainedwith dispase.

FIG. 22: Experimental 1 of testing duration of H₁₁₅₂ and aggregateformation.

FIG. 23: Experimental 2 of testing duration of H₁₁₅₂ and aggregateformation.

FIGS. 24A-J: Images from Experiment 1 showing differences in aggregatenumbers on day 14 of differentiation. FIG. 24A: 3e5 cells/ml initialseeding with 1 day H₁₁₅₂. FIG. 24B: 3e5 cells/ml initial seeding with 2days H₁₁₅₂. FIG. 24C: 5e5 cells/ml initial seeding with 1 day H₁₁₅₂.FIG. 24D: 5e5 cells/ml initial seeding with 2 days H₁₁₅₂. FIG. 24E: 7e5cells/ml initial seeding with 1 day H₁₁₅₂. FIG. 24F: 7e5 cells/mlinitial seeding with 2 days H₁₁₅₂. FIG. 24G: 10e5 cells/ml initialseeding with 1 day H₁₁₅₂. FIG. 24H: 10 e 5 cells/ml initial seeding with2 days H₁₁₅₂. FIG. 241: 15 e 5 cells/ml initial seeding with 1 dayH₁₁₅₂. FIG. 24J: 15e5 cells/ml initial seeding with 2 days H₁₁₅₂.

FIG. 25: Repeated results to show benefits of H₁₁₅₂ for cardiac cellproduction in conditions containing H₁₁₅₂ for 48 hours.

FIG. 26: Plot of final cell counts and purity from Experiment 1suggesting that prolonged H₁₁₅₂ exposure impacts purity via celldensity, not through an independent mechanism.

FIG. 27: Plot of final cell counts and purity from Experiment 2suggesting that prolonged H₁₁₅₂ exposure impacts purity via celldensity, not through an independent mechanism.

FIG. 28: Effect of different concentrations of H₁₁₅₂ on cell survivalwith one day dose of H₁₁₅₂ on IPS 6.1 cells.

FIGS. 29A-B: Cardiac induction of iPS cells in the presence of variousconcentration of HGF and FGF. Cardiomyocyte yield and iPS cellconversion efficiency ratio as a function of HGF concentration. Errorbars represent SEM (standard error of the mean) for n=3 T25 flask s inone experiment.

FIGS. 30A-B: Cardiac induction of iPS cells in the presence of variousconcentration of HGF and FGF. Error bars represent SEM for n=3 T25 flasks in one experiment.

FIGS. 31A-C: Cardiomyocyte yield (FIG. 31A), H9-TGZ cell conversionefficiency ratio (FIG. 31B), and CM purity (FIG. 31C) as a function ofHGF concentration. Each point represents a single T25 flask consolidatedfrom multiple T25 flasks on day 2 due to low aggregate formationefficiency.

FIGS. 32A-C: Cardiomyocyte yield (FIG. 32A), H9-TGZ cell conversionefficiency ratio (FIG. 32B), and CM purity (FIG. 32C) as a function ofHGF concentration. Error bars represent SEM for n=3 T25 flasks in oneexperiment.

FIGS. 33A-D: The results of a growth factor/inhibitor screen utilizingvarying concentrations of BMP (1 ng/mL, 10 ng/mL), dorsomorphin (2 uM,0.2 uM), Activin A (1 ng/mL, 10 ng/mL), SB-431542 (10 uM, 0.1 uM) and acombination of Activin A (6 ng/mL) and BMP4 (10 ng/mL). FIG. 33C isactual data for 15 ml culture and FIG. 33D represents the calculated 1 Lscale-up (as a projection for production) based on FIG. 33C data.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. METHODS AND COMPOSITIONS

A variety of different methods and compositions are described herein.Certain embodiments concern several important advantages that improvethe cardiomyocyte production process. First of all, it has beendiscovered that aggregate size can be controlled in a suspensionculture, which provides optimal conditions for cardiac induction, suchas through optimal rotary speed, seeding density, and/or time untilcardiac induction. In some embodiments such methods increase uniformityand yields of the differentiated cells. Secondly, ROCK inhibitors havebeen combined with suspension culture to improve cell survival anddifferentiation in large volume culture vessels, such as bioreactors. Insome embodiments, the concentration and/or duration of ROCK inhibitorincubation has been optimized. Further, provided herein is an exemplarytechnique for determining the appropriate adjustments in growth factoradditions that can be employed to dramatically improve cardiacdifferentiation for any given medium batch or pluripotent cell cloneemployed.

Further advances in the production of cardiomyocyte cell populations arealso described below. The remarkable uniformity and functionalproperties of the cells produced according to this disclosure make themvaluable for studying cardiac tissue in vitro, and for developing newtherapeutic modalities for regeneration of cardiac tissue in thetreatment of heart disease.

II. DEFINITIONS

“Pluripotency” refers to a stem cell that has the potential todifferentiate into all cells constituting one or more tissues or organs,for example, any of the three germ layers: endoderm (interior stomachlining, gastrointestinal tract, the lungs), mesoderm (muscle, bone,blood, urogenital), or ectoderm (epidermal tissues and nervous system).“Pluripotent stem cells” (or PSCs) used herein refer to cells that candifferentiate into cells derived from any of the three germ layers, forexample, descendants of totipotent cells, embryonic stem cells, orinduced pluripotent stem cells.

“Induced pluripotent stem cells,” commonly abbreviated as iPS cells oriPSCs, refer to a type of pluripotent stem cell artificially preparedfrom a non-pluripotent cell, typically an adult somatic cell, orterminally differentiated cell, such as fibroblast, a hematopoieticcell, a myocyte, a neuron, an epidermal cell, or the like, byintroducing or contacting reprogramming factors.

“Embryonic stem (ES) cells” are pluripotent stem cells derived fromearly embryos.

“Suspension culture,” refers to a culture in which cells, or aggregatesof cells, multiply while suspended in liquid medium.

“Rho-associated kinase inhibitors,” abbreviated as “ROCK inhibitors,”refer to any substance that inhibits or reduces the function ofRho-associated kinase or its signaling pathway in a cell, such as asmall molecule, an siRNA, a miRNA, an antisense RNA, or the like. “ROCKsignaling pathway,” as used herein, may include any signal processorsinvolved in the ROCK-related signaling pathway, such as theRho-ROCK-Myosin II signaling pathway, its upstream signaling pathway, orits downstream signaling pathway in a cell. Examples of ROCK inhibitorsinclude, but are not limited to, a Rho-specific inhibitor, aROCK-specific inhibitor, a MRLC (myosin regulatory light chain)-specificinhibitor, or a Myosin II-specific inhibitor.

The term “aggregate promoting medium” means any medium that enhances theaggregate formation of stem cells without any restriction as to the modeof action.

The term “cardiac induction medium” or “cardiac differentiation medium”means any medium that enhances the differentiation of stem cells tocardiomyocytes without any restriction as to the mode of action.

The term “cardiac maintenance medium” means any medium that is suitablefor maintenance of cardiomyocytes without any restriction as to the modeof action.

The term “aggregates,” i.e., embryoid bodies, refers to heterogeneousclusters comprising differentiated and partly differentiated cells thatappear when pluripotent stem cells are allowed to differentiate in anon-specific fashion.

“Cardiomyocytes” refers generally to any cardiomyocytes lineage cells,and can be taken to apply to cells at any stage of cardiomyocytesontogeny without any restriction, unless otherwise specified. Forexample, cardiomyocytes may include both cardiomyocyte precursor cellsand mature cardiomyocytes.

A “gene,” “polynucleotide,” “coding region,” “sequence, ” “segment,” or“fragment,” which “encodes” a particular protein, is a nucleic acidmolecule which is transcribed and optionally also translated into a geneproduct, e.g., a polypeptide, in vitro or in vivo when placed under thecontrol of appropriate regulatory sequences. The coding region may bepresent in either a cDNA, genomic DNA, or RNA form. When present in aDNA form, the nucleic acid molecule may be single-stranded (i.e., thesense strand) or double-stranded. The boundaries of a coding region aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A gene can include, but is notlimited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the gene sequence.

The term “transgene,” refers to a gene, nucleic acid, or polynucleotidewhich has been introduced into the cell or organism by artificial ornatural means, such as an exogenous nucleic acid. An exogenous nucleicacid may be from a different organism or cell, or it may be one or moreadditional copies of a nucleic acid which occurs naturally within theorganism or cell. By way of a non-limiting example, an exogenous nucleicacid is in a chromosomal location different from that of natural cells,or is otherwise flanked by a different nucleic acid sequence than thatfound in nature.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream (3′direction) coding sequence.

III. SOURCES OF PLURIPOTENT STEM CELLS

The term “pluripotent stem cell” refers to a cell capable of giving riseto cells of all three germinal layers, that is, endoderm, mesoderm andectoderm. Although in theory a pluripotent stem cell can differentiateinto any cell of the body, the experimental determination ofpluripotency is typically based on differentiation of a pluripotent cellinto several cell types of each germinal layer. In some embodiments ofthe present invention, a pluripotent stem cell is an embryonic stem (ES)cell derived from the inner cell mass of a blastocyst. In otherembodiments, the pluripotent stem cell is an induced pluripotent stemcell derived by reprogramming somatic cells. In certain embodiments, thepluripotent stem cell is an embryonic stem cell derived by somatic cellnuclear transfer.

A. Embryonic Stem Cells

Embryonic stem (ES) cells are pluripotent cells derived from the innercell mass of a blastocyst. ES cells can be isolated by removing theouter trophectoderm layer of a developing embryo, then culturing theinner mass cells on a feeder layer of non-growing cells. Underappropriate conditions, colonies of proliferating, undifferentiated EScells are produced. The colonies can be removed, dissociated intoindividual cells, then replated on a fresh feeder layer. The replatedcells can continue to proliferate, producing new colonies ofundifferentiated ES cells. The new colonies can then be removed,dissociated, replated again and allowed to grow. This process of“subculturing” or “passaging” undifferentiated ES cells can be repeateda number of times to produce cell lines containing undifferentiated EScells (U.S. Pat. Nos. 5,843,780; 6,200,806; 7,029,913). A “primary cellculture” is a culture of cells directly obtained from a tissue such asthe inner cell mass of a blastocyst. A “subculture” is any culturederived from the primary cell culture.

Methods for obtaining mouse ES cells are well known. In one method, apreimplantation blastocyst from the 129 strain of mice is treated withmouse antiserum to remove the trophoectoderm, and the inner cell mass iscultured on a feeder cell layer of chemically inactivated mouseembryonic fibroblasts in medium containing fetal calf serum. Colonies ofundifferentiated ES cells that develop are subcultured on mouseembryonic fibroblast feeder layers in the presence of fetal calf serumto produce populations of ES cells. In some methods, mouse ES cells canbe grown in the absence of a feeder layer by adding the cytokineleukemia inhibitory factor (LIF) to serum-containing culture medium(Smith, 2000). In other methods, mouse ES cells can be grown inserum-free medium in the presence of bone morphogenetic protein and LIF(Ying et al., 2003).

Human ES cells can be obtained from blastocysts using previouslydescribed methods (Thomson et al., 1995; Thomson et al., 1998; Thomsonand Marshall, 1998; Reubinoff et al, 2000.) In one method, day-5 humanblastocysts are exposed to rabbit anti-human spleen cell antiserum, thenexposed to a 1:5 dilution of Guinea pig complement to lyse trophectodermcells. After removing the lysed trophectoderm cells from the intactinner cell mass, the inner cell mass is cultured on a feeder layer ofgamma-inactivated mouse embryonic fibroblasts and in the presence offetal bovine serum. After 9 to 15 days, clumps of cells derived from theinner cell mass can be chemically (i.e. exposed to trypsin) ormechanically dissociated and replated in fresh medium containing fetalbovine serum and a feeder layer of mouse embryonic fibroblasts. Uponfurther proliferation, colonies having undifferentiated morphology areselected by micropipette, mechanically dissociated into clumps, andreplated (see U.S. Pat. No. 6,833,269). ES-like morphology ischaracterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells can beroutinely passaged by brief trypsinization or by selection of individualcolonies by micropipette. In some methods, human ES cells can be grownwithout serum by culturing the ES cells on a feeder layer of fibroblastsin the presence of basic fibroblast growth factor (Amit et al., 2000).In other methods, human ES cells can be grown without a feeder celllayer by culturing the cells on a protein matrix such as Matrigel™ orlaminin in the presence of “conditioned” medium containing basicfibroblast growth factor (Xu et al., 2001). The medium is previouslyconditioned by coculturing with fibroblasts.

Methods for the isolation of rhesus monkey and common marmoset ES cellsare also known (Thomson, and Marshall, 1998; Thomson et al., 1995;Thomson and Odorico, 2000).

Another source of ES cells are established ES cell lines. Various mousecell lines and human ES cell lines are known and conditions for theirgrowth and propagation have been defined. For example, the mouse CGR8cell line was established from the inner cell mass of mouse strain 129embryos, and cultures of CGR8 cells can be grown in the presence of LIFwithout feeder layers. As a further example, human ES cell lines H1, H7,H9, H13 and H14 were established by Thompson et al. In addition,subclones H9.1 and H9.2 of the H9 line have been developed. It isanticipated that virtually any ES or stem cell line known in the art andmay be used with the present invention, such as, e.g., those describedin Yu and Thompson, 2008, which is incorporated herein by reference.

The source of ES cells for use in connection with the present inventioncan be a blastocyst, cells derived from culturing the inner cell mass ofa blastocyst, or cells obtained from cultures of established cell lines.Thus, as used herein, the term “ES cells” can refer to inner cell masscells of a blastocyst, ES cells obtained from cultures of inner masscells, and ES cells obtained from cultures of ES cell lines.

A pluripotent cell is capable of differentiating into any cell of thebody. The pluripotency of ES cells has been determined in various ways(Martin, 1982). In one test, mouse ES cells derived from the inner cellmass of a blastocyst are injected into the cavity of another blastocyst.The injected blastocyst is deposited into the uterus of a pseudopregnantfemale mouse to produce progeny that are chimeras of injected andrecipient blastocyst cells. In another test, mouse ES cells are injectedinto adult mice to produce tumors called teratomas. Such tumors cancontain a variety of cell types derived from endoderm, mesoderm, andectoderm. In certain embodiments, one or more teratoma-derived cells maybe cultured or differentiated into neuronal or neuronal-committed cells.The pluripotency of human ES cells can also be tested by the formationof teratomas in immunodeficient mice. A third test is to alter cultureconditions to allow ES cells to differentiate into more specializedcells. For example, mouse ES cells can spontaneously differentiate intovarious cell types by removing the feeder layer and adding LIF to theculture medium. Similarly, human ES cells can spontaneouslydifferentiate by removing the feeder layer and growing the ES cells on anon-adherent surface in suspension (Itskovitz-Eldor et al., 2000;Reubinoff et al., 2000; Roach et al., 1993). Under such conditions, theES cells can form cell aggregates called embryoid bodies which containcells having characteristics of neurons and heart muscle cells. In allof these tests, the pluripotency of ES cells is shown by their abilityto generate cells of endoderm, mesoderm, and ectoderm origin.

ES cells can be characterized by the proteins they produce. For example,the following marker proteins have been used to characterize ES cells:stage-specific embryonic antigen SSEA-1, stage-specific embryonicantigen SSEA-3, stage-specific embryonic antigen SSEA-4, tumor rejectionantigen-1-60 (TRA1-60), tumor rejection antigen-1-81 (TRA1-81), alkalinephosphatase (AP), and transcription factor Oct-4. As shown in Table 1,mouse, human and primate cells differ in their pattern of expression ofthese markers. For example, SSEA-1 is expressed in mouse ES cells, butnot human or monkey ES cells, while TRA1-60 is expressed in human andmonkey ES cells but not mouse ES cells.

TABLE 1 ES Cell Marker Expression Marker Mouse Human Monkey SSEA-1 YesNo No SSEA-2 No Yes Yes SSEQ-3 No Yes Yes TRA1-60 No Yes Yes TRA1-81 NoYes Yes AP Yes Yes Yes Oct-4 Yes Yes Yes

B. Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells are cells which have thecharacteristics of ES cells but are obtained by the reprogramming ofdifferentiated somatic cells. Induced pluripotent stem cells have beenobtained by various methods. In one method, adult human dermalfibroblasts are transfected with transcription factors Oct4, Sox2, c-Mycand Klf4 using retroviral transduction (Takahashi et al., 2007). Thetransfected cells are plated on SNL feeder cells (a mouse cellfibroblast cell line that produces LIF) in medium supplemented withbasic fibroblast growth factor (bFGF). After approximately 25 days,colonies resembling human ES cell colonies appear in culture. The EScell-like colonies are picked and expanded on feeder cells in thepresence of bFGF.

Based on cell characteristics, cells of the ES cell-like colonies areinduced pluripotent stem cells. The induced pluripotent stem cells aremorphologically similar to human ES cells, and express various human EScell markers. Also, when grown under conditions that are known to resultin differentiation of human ES cells, the induced pluripotent stem cellsdifferentiate accordingly. For example, the induced pluripotent stemcells can differentiate into cells having neuronal structures andneuronal markers. It is anticipated that virtually any iPS cells or celllines may be used with the present invention, including, e.g., thosedescribed in Yu and Thompson, 2008.

In another method, human fetal or newborn fibroblasts are transfectedwith four genes, Oct4, Sox2, Nanog and Lin28 using lentivirustransduction (Yu et al., 2007). At 12-20 days post infection, colonieswith human ES cell morphology become visible. The colonies are pickedand expanded. The induced pluripotent stem cells making up the coloniesare morphologically similar to human ES cells, express various human EScell markers, and form teratomas having neural tissue, cartilage and gutepithelium after injection into mice.

Methods of preparing induced pluripotent stem cells from mouse are alsoknown (Takahashi and Yamanaka, 2006). Induction of iPS cells typicallyrequire the expression of or exposure to at least one member from Soxfamily and at least one member from Oct family. Sox and Oct are thoughtto be central to the transcriptional regulatory hierarchy that specifiesES cell identity. For example, Sox may be Sox-1, Sox-2, Sox-3, Sox-15,or Sox-18; Oct may be Oct-4. Additional factors may increase thereprogramming efficiency, like Nanog, Lin28, Klf4, or c-Myc; specificsets of reprogramming factors may be a set comprising Sox-2, Oct-4,Nanog and, optionally, Lin-28; or comprising Sox-2, Oct4, Klf and,optionally, c-Myc.

IPS cells, like ES cells, have characteristic antigens that can beidentified or confirmed by immunohistochemistry or flow cytometry, usingantibodies for SSEA-1, SSEA-3 and SSEA-4 (Developmental StudiesHybridoma Bank, National Institute of Child Health and HumanDevelopment, Bethesda Md.), and TRA-1-60 and TRA-1-81 (Andrews et al.,1987). Pluripotency of embryonic stem cells can be confirmed byinjecting approximately 0.5-10×10⁶ cells into the rear leg muscles of8-12 week old male SCID mice. Teratomas develop that demonstrate atleast one cell type of each of the three germ layers.

In certain aspects of the present invention, iPS cells are maded fromreprogramming somatic cells using reprogramming factors comprising Octfamily member and a Sox family member, such as Oct4 and Sox2 incombination with Klf or Nanog as describe above. The somatic cell in thepresent invention may be any somatic cell that can be induced topluripotency, such as a fibroblast, a keratinocyte, a hematopoieticcell, a mesenchymal cell, a liver cell, a stomach cell, or a β cell. Ina certain aspect, T cells may also be used as source of somatic cellsfor reprogramming (see U.S. Application No. 61/184,546, incorporatedherein by reference).

Reprogramming factors may be expressed from expression cassettescomprised in one or more vectors, such as an integrating vector or anepisomal vector, such as a EBV element-based system (see U.S.Application No. 61/058,858, incorporated herein by reference; Yu et al.,2009). In a further aspect, reprogramming proteins could be introduceddirectly into somatic cells by protein transduction (see U.S.Application No. 61/172,079, incorporated herein by reference).

C. Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Pluripotent stem cells can be prepared by means of somatic cell nucleartransfer, in which a donor nucleus is transferred into a spindle-freeoocyte. Stem cells produced by nuclear transfer are geneticallyidentical to the donor nuclei. In one method, donor fibroblast nucleifrom skin fibroblasts of a rhesus macaque are introduced into thecytoplasm of spindle-free, mature metaphase II rhesus macaque ooctyes byelectrofusion (Byrne et al., 2007). The fused oocytes are activated byexposure to ionomycin, then incubated until the blastocyst stage. Theinner cell mass of selected blastocysts are then cultured to produceembryonic stem cell lines. The embryonic stem cell lines show normal EScell morphology, express various ES cell markers, and differentiate intomultiple cell types both in vitro and in vivo. As used herein, the term“ES cells” refers to embryonic stem cells derived from embryoscontaining fertilized nuclei. ES cells are distinguished from embryonicstem cells produced by nuclear transfer, which are referred to as“embryonic stem cells derived by somatic cell nuclear transfer.”

IV. CULTURING OF PLURIPOTENT STEM CELLS

Depending on culture conditions, pluripotent stem cells can producecolonies of differentiated cells or undifferentiated cells. The term“differentiate” means the progression of a cell down a developmentalpathway. The term “differentiated” is a relative term describing acell's progression down a developmental pathway in comparison withanother cell. For example, a pluripotent cell can give rise to any cellof the body, while a more differentiated cell such as a hematopoeticcell will give rise to fewer cell types.

Cultures of pluripotent stem cells are described as “undifferentiated”when a substantial proportion of stem cells and their derivatives in thepopulation display morphological characteristics of undifferentiatedcells, clearly distinguishing them from differentiated cells of embryoor adult origin. Undifferentiated ES or iPS cells are recognized bythose skilled in the art, and typically appear in the two dimensions ofa microscopic view in colonies of cells with high nuclear/cytoplasmicratios and prominent nucleoli. It is understood that colonies ofundifferentiated cells can have neighboring cells that aredifferentiated.

In certain aspects, starting cells for the present methods may compriseat least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³cells or any range derivable therein. The starting cell population mayhave a seeding density of at least or about 10, 10¹, 10², 10³, 10⁴, 10⁵,10⁶, 10⁷, 10⁸ cells/ml, or any range derivable therein.

A. Culturing of ES cells

ES cells can be maintained in an undifferentiated state by culturing thecells in the presence of serum and a feeder layer, typically mouseembryonic fibroblasts. Other methods for maintaining stem cells in anundifferentiated state are also known. For example, mouse ES cells canbe maintained in an undifferentiated state by culturing in the presenceof LIF without a feeder layer. However, unlike mouse ES cells, human EScells do not respond to LIF. Human ES cells can be maintained in anundifferentiated state by culturing ES cells on a feeder layer offibroblasts in the presence of basic fibroblast growth factor (Amit etal., 2000), or by culturing on a protein matrix, such as Matrigel™ orlaminin, without a feeder layer and in the presence offibroblast-conditioned medium plus basic fibroblast growth factor, (Xuet al., 2001; U.S. Pat. No. 6,833,269).

Methods for preparing and culturing ES cells can be found in standardtextbooks and reviews in cell biology, tissue culture, and embryology,including teratocarcinomas and embryonic stem cells: A practicalapproach (1987); Guide to Techniques in Mouse Development (1993);Embryonic Stem Cell Differentiation in vitro (1993); Properties and usesof Embryonic Stem Cells: Prospects for Application to Human Biology andGene Therapy (1998), all incorporated herein by reference. Standardmethods used in tissue culture generally are described in Animal CellCulture (1987); Gene Transfer Vectors for Mammalian Cells (1987); andCurrent Protocols in Molecular Biology and Short Protocols in MolecularBiology (1987 & 1995).

B. Culturing of iPS Cells

After somatic cells are introduced or contacted with reprogrammingfactors, these cells may be cultured in a medium sufficient to maintainthe pluripotency and the undifferentiated state. Culturing of inducedpluripotent stem (iPS) cells generated in this invention can use variousmedium and techniques developed to culture primate pluripotent stemcells, more specially, embryonic stem cells, as described in U.S. Pat.Publication 20070238170 and U.S. Pat. Publication 20030211603, and U.S.Pat. Publication 20080171385, which are hereby incorporated byreference. It is appreciated that additional methods for the culture andmaintenance of pluripotent stem cells, as would be known to one ofskill, may be used with the present invention.

In certain embodiments, undefined conditions may be used; for example,pluripotent cells may be cultured on fibroblast feeder cells or a mediumthat has been exposed to fibroblast feeder cells in order to maintainthe stem cells in an undifferentiated state. Alternately, pluripotentcells may be cultured and maintained in an essentially undifferentiatedstate using defined, feeder-independent culture system, such as a TeSRmedium (Ludwig et al., 2006a; Ludwig et al., 2006b). Feeder-independentculture systems and media may be used to culture and maintainpluripotent cells. These approaches allow human embryonic stem cells toremain in an essentially undifferentiated state without the need formouse fibroblast “feeder layers.” As described herein, variousmodifications may be made to these methods in order to reduce costs asdesired.

Various matrix components may be used in culturing and maintaining humanpluripotent stem cells. For example, collagen IV, fibronectin, laminin,and vitronectin in combination may be used to coat a culturing surfaceas a means of providing a solid support for pluripotent cell growth, asdescribed in Ludwig et al. (2006a; 2006b), which are incorporated byreference in their entirety.

Matrigel™ may also be used to provide a substrate for cell culture andmaintenance of human pluripotent stem cells. Matrigel™ is a gelatinousprotein mixture secreted by mouse tumor cells and is commerciallyavailable from BD Biosciences (New Jersey, USA). This mixture resemblesthe complex extracellular environment found in many tissues and is usedby cell biologists as a substrate for cell culture.

C. ROCK Inhibitors and Myosin II ATPase Inhibitors

Pluripotent stem cells, especially human ES cells and iPS cells, arevulnerable to apoptosis upon cellular detachment and dissociation, whichare important for clonal isolation or expansion and differentiationinduction. Recently, a small class of molecules have been found toincrease clonal efficiency and survival of dissociated pluripotent stemcells, such as Rho-associated kinase (ROCK) inhibitors, which areinhibitors for ROCK-related signaling pathways, for example,Rho-specific inhibitors, ROCK-specific inhibitors or myosin II-specificinhibitors. In certain aspects of the invention, ROCK inhibitors may beused for culturing and passaging of pluripotent stem cells and/ordifferentiation of the stem cells. Therefore, ROCK inhibitors could bepresent in any cell culture medium in which pluripotent stem cells grow,dissociate, form aggregates, or undergo differentiation, such as anadherent culture or suspension culture.

ROCK signaling pathways may include Rho family GTPases, ROCK, a majoreffector kinase downstream of Rho, Myosin II, the predominant effectordownstream of ROCK (Harb et al., 2008), and any intermediate, upstream,or downstream signal processors. ROCK may phosphorylate and inactivatemyosin phosphatase target subunit 1 (MYPT1), one of the major downstreamtargets of ROCK that negatively regulates myosin function throughdephosphorylation of myosin regulatory light chain (MRLC).

Rho-specific inhibitors, such as Clostridium botulinum C3 exoenzyme,and/or Myosin II-specific inhibitors may also be used as a ROCKinhibitor in certain aspects of the invention. Unless otherwise statedherein, myosin II inhibitors, such as blebbistatin, can substitute forthe experimental use of ROCK inhibitors.

Myosin II was first studied for its role in muscle contraction, but itfunctions also in non-muscle cells. Myosin II (also known asconventional myosin) contains two heavy chains, each about 2000 aminoacids in length, which constitute the head and tail domains. Each ofthese heavy chains contains the N-terminal head domain, while theC-terminal tails take on a coiled-coil morphology, holding the two heavychains together (imagine two snakes wrapped around each other, such asin a caduceus). Thus, myosin II has two heads. It also contains 4 lightchains (2 per head), which bind the heavy chains in the “neck” regionbetween the head and tail. These light chains are often referred to asthe essential light chain and the regulatory light chain. An exemplaryMyosin II-specific inhibitor may be Blebbistatin or its derivatives.

ROCKs are serine/threonine kinases that serve as a target proteins forRho (of which three isoforms exist—RhoA, RhoB and RhoC). Theses kinaseswere initially characterized as mediators of the formation ofRhoA-induced stress fibers and focal adhesions. The two ROCKisoforms—ROCK1 (p160ROCK, also called ROKβ) and ROCK2 (ROKα)—arecomprised of a N-terminal kinase domain, followed by a coiled-coildomain containing a Rho-binding domain and a pleckstrin-homology domain(PH). Both ROCKs are cytoskeletal regulators, mediating RhoA effects onstress fiber formation, smooth muscle contraction, cell adhesion,membrane ruffling and cell motility. ROCKs may exert their biologicalactivity by targeting downstream molecules, such as myosin II, myosinlight chain (MLC), MLC phosphatase (MLCP) and the phosphatase and tensinhomolog (PTEN).

An exemplary ROCK-specific inhibitor is Y-27632, which selectivelytargets ROCK1 (but also inhibits ROCK2), as well as inhibits TNF-α andIL-β. It is cell permeable and inhibits ROCK1/ROCK2 (IC₅₀=800 nM) bycompeting with ATP. Ishizaki et al. (2000), incorporated herein byreference as if set forth in its entirety. Other ROCK inhibitorsinclude, e.g., H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077,GSK269962A and SB-772077-B. Doe et al. (2007); Ishizaki et al., supra;Nakajima et al. (2003); and Sasaki et al. (2002), each of which isincorporated herein by reference as if set forth in its entirety.

Other non-limiting examples of ROCK inhibitors include H-1152 andFasudil (also referred to as HA1077), Y-30141 (described in U.S. Pat.No. 5,478,838), and derivatives thereof, and antisense nucleic acid forROCK, RNA interference inducing nucleic acid (for example, siRNA),competitive peptides, antagonist peptides, inhibitory antibodies,antibody-ScFV fragments, dominant negative variants and expressionvectors thereof. Further, since other low molecular compounds are knownas ROCK inhibitors, such compounds or derivatives thereof can be alsoused in embodiments (for example, refer to U.S. Patent Publication Nos.20050209261, 20050192304, 20040014755, 20040002508, 20040002507,20030125344 and 20030087919, and International Patent Publication Nos.2003/062227, 2003/059913, 2003/062225, 2002/076976 and 2004/039796,which are hereby incorporated by reference). In the present invention, acombination of one or two or more of the ROCK inhibitors can also beused.

According to some embodiments, the stem cell can be treated with a ROCKinhibitor in a medium. Thereby, the medium used in the methods of thepresent invention may already contain the ROCK inhibitor oralternatively, the methods of the present invention may involve a stepof adding the ROCK inhibitor to the medium. The concentration of theROCK inhibitor in the medium is particularly not limited as far as itcan achieve the desired effects such as the improved survival rate ofstem cells. Such a ROCK inhibitor, e.g., Y-27632, HA-1077, or H-1152,may be used at an effective concentration of at least or about 0.02,0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 150, 200, 500 to about 1000 μM, or any range derivable therein.These amounts may refer to an amount of a ROCK inhibitor individually orin combination with one or more ROCK inhibitors.

For example, when Y-27632 is used as the ROCK inhibitor, it can be usedat the concentration of about 0.01 to about 1000 μM, more specificallyabout 0.1 to about 100 μM, further more specifically about 1.0 to about30 μM, and most specifically about 2.0 to 20 μM, or any range derivabletherein. When Fasudil/HA1077 is used as the ROCK inhibitor, it can beused at about twofold the aforementioned Y-27632 concentration. WhenH-1152 is used as the ROCK inhibitor, it can be used at about 1/50th ofthe aforementioned Y-27632 concentration.

The time for treating with the ROCK inhibitor is particularly notlimited as long as it is a time duration for which the desired effectssuch as the improved survival rate of stem cells can be achieved. Forexample, when the stem cell is a pluripotent stem cells such as a humanembryonic stem cell, the time for treating is at least or about 10, 15,20, 25, 30 minutes to several hours (e.g., at least or about one hour,two hours, three hours, four hours, five hours, six hours, eight hours,12 hours, 16 hours, 24 hours, 36 hours, 48 hours, or any range derivabletherein) before dissociation. After dissociation, the pluripotent stemcell can be treated with the ROCK inhibitor for, for example, at leastor about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 24, 48 hours or more toachieve the desired effects.

The density of the stem cell(s) to be treated with the ROCK inhibitor isparticularly not limited as far as it is a density at which the desiredeffects such as the improved survival rate of stem cells can beachieved. It is, for example, about 1.0×10¹ to 1.0×10⁷ cells/ml, moreparticularly about 1.0×10² to 1.0×10′ cells/ml, further moreparticularly about 1.0×10³ to 1.0×10⁷ cells/ml, and most particularlyabout 3.0×10⁴ to 2.0×10⁶ cells/ml.

In certain embodiments, stem cells are cultured in the presence of ROCKinhibitors to improve survival at low density (dissociated into singlecells or small aggregates), cloning efficiency or passaging efficiency.In certain embodiments of the invention, the stem cells are cultured inthe absence of feeder cells, feeder cell extracts and/or serum. The stemcells can be cultured in the presence of a ROCK inhibitor prior tosubcloning or passaging, e.g., for at least one hour before subcloningor passaging. Alternatively or additionally, the stem cells aremaintained in the presence of a ROCK inhibitor during or aftersubcloning or passaging.

In certain embodiments, the stem cells are maintained in the presence ofa ROCK inhibitor for at most or at least about 4, 8, 12 hours, about 2,about 4, or about 6 days, or any range derivable therein. In otherembodiments, the stem cells are maintained in the presence of a ROCKinhibitor for at least one to five passages. Optionally, the ROCKinhibitor is subsequently withdrawn from the culture medium, for exampleafter about 4, 8, 12 hours or after about 2, about 4, or about 6 days,or any range derivable therein. In other embodiments, the ROCK inhibitoris withdrawn after at least one, two, three, four, five passages ormore, or any range derivable therein.

The stem cells to be treated with a ROCK inhibitor according to thepresent invention can be dissociated cells or non-dissociated cells. Thedissociated cells refer to cells treated to promote cell dissociation(for example, the dissociation described later). Dissociated cellsinclude a single cell and cells having formed a small cell clump(aggregate) of several (typically about 2 to 50, 2 to 20, or 2 to 10)cells. The dissociated cells can be suspended (floating) cells oradhered cells. For example, it has been known that ES cells such ashuman ES cells are susceptible to specific conditions such asdissociation (and/or suspension culture after dissociation). The methodsof the present invention have particular use when the stem cell issubject to conditions at which hitherto cell death would have occurred.

Certain aspects of the present invention can further involve a step ofdissociating stem cells. Stem cell dissociation can be performed usingany known procedures. These procedures include treatments with achelating agent (such as EDTA), an enzyme (such as trypsin,collagenase), or the like, and operations such as mechanicaldissociation (such as pipetting). The stem cell(s) can be treated withthe ROCK inhibitor before and/or after dissociation. For example, thestem cell(s) can be treated only after dissociation.

D. Stem Cell Culture Conditions

The culturing conditions according to the present invention will beappropriately defined depending on the medium and stem cells used. Themedium according to the present invention can be prepared using a mediumto be used for culturing animal cells as its basal medium. As the basalmedium, any of TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEMZinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI 1640,and Fischer's media, as well as any combinations thereof can be used,but the medium is not particularly limited thereto as far as it can beused for culturing animal cells.

The medium according to the present invention can be a serum-containingor serum-free medium. The serum-free medium refers to media with nounprocessed or unpurified serum and accordingly, can include media withpurified blood-derived components or animal tissue-derived components(such as growth factors). From the aspect of preventing contaminationwith heterogeneous animal-derived components, serum can be derived fromthe same animal as that of the stem cell(s).

The medium according to the present invention may contain or may notcontain any alternatives to serum. The alternatives to serum can includematerials which appropriately contain albumin (such as lipid-richalbumin, albumin substitutes such as recombinant albumin, plant starch,dextrans and protein hydrolysates), transferrin (or other irontransporters), fatty acids, insulin, collagen precursors, traceelements, 2-mercaptoethanol, 3′-thiolgiycerol, or equivalents thereto.The alternatives to serum can be prepared by the method disclosed inInternational Publication No. 98/30679, for example. Alternatively, anycommercially available materials can be used for more convenience. Thecommercially available materials include knockout Serum Replacement(KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax(Gibco).

The medium of the present invention can also contain fatty acids orlipids, amino acids (such as non-essential amino acids), vitamin(s),growth factors, cytokines, antioxidant substances, 2-mercaptoethanol,pyruvic acid, buffering agents, and inorganic salts. The concentrationof 2-mercaptoethanol can be, for example, about 0.05 to 1.0 mM, andparticularly about 0.1 to 0.5 mM, but the concentration is particularlynot limited thereto as long as it is appropriate for culturing the stemcell(s).

A culture vessel used for culturing the stem cell(s) can include, but isparticularly not limited to: flask, flask for tissue culture, dish,petri dish, dish for tissue culture, multi dish, micro plate, micro-wellplate, multi plate, multi-well plate, micro slide, chamber slide, tube,tray, CellSTACK® Chambers, culture bag, and roller bottle, as long as itis capable of culturing the stem cells therein. The stem cells may beculture in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30,40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any rangederivable therein, depending on the needs of the culture. In a certainembodiment, the culture vessel may be a bioreactor, which may refer toany device or system that supports a biologically active environment.The bioreactor may have a volume of at least or about 2, 4, 5, 6, 8, 10,15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15cubic meters, or any range derivable therein.

The culture vessel can be cellular adhesive or non-adhesive and selecteddepending on the purpose. The cellular adhesive culture vessel can becoated with any of substrates for cell adhesion such as extracellularmatrix (ECM) to improve the adhesiveness of the vessel surface to thecells. The substrate for cell adhesion can be any material intended toattach stem cells or feeder cells (if used). The substrate for celladhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine,laminin, and fibronectin and mixtures thereof for example Matrigel™, andlysed cell membrane preparations (Klimanskaya et al., 2005).

Other culturing conditions can be appropriately defined. For example,the culturing temperature can be about 30 to 40° C., for example, atleast or about 31, 32, 33, 34, 35, 36, 37, 38, 39° C. but particularlynot limited to them. The CO₂ concentration can be about 1 to 10%, forexample, about 2 to 5%, or any range derivable therein. The oxygentension can be at least or about 1, 5, 8, 10, 20%, or any rangederivable therein.

The methods of the present invention can be used for adhesion culture ofstem cells, for example. In this case, the cells can be cultured in thepresence of feeder cells. In the case where the feeder cells are used inthe methods of the present invention, stromal cells such as fetalfibroblasts can be used as feeder cells (for example, refer to;Manipulating the Mouse Embryo A Laboratory Manual (1994); GeneTargeting, A Practical Approach (1993); Martin (1981); Evans et al.(1981); Jainchill et al., (1969); Nakano et al., Science (1996); Kodamaet al. (1982); and International Publication Nos. 01/088100 and2005/080554).

The methods of the present invention can be also used for a suspensionculture of stem cells, including suspension culture on carriers(Fernandes et al., 2007) or gel/biopolymer encapsulation (United StatesPatent 20070116680). The term suspension culture of the stem cells meansthat the stem cells are cultured under non-adherent condition withrespect to the culture vessel or feeder cells (if used) in a medium. Thesuspension culture of stem cells includes a dissociation culture of stemcells and an aggregate suspension culture of stem cells. The termdissociation culture of stem cells means that suspended stem cells iscultured, and the dissociation culture of stem cells include those ofsingle stem cell or those of small cell aggregates composed of aplurality of stem cells (for example, about 2 to 400 cells). When theaforementioned dissociation culture is continued, the cultured,dissociated cells form a larger aggregate of stem cells, and thereafteran aggregate suspension culture can be performed. The aggregatesuspension culture includes an embryoid culture method (see Keller etal., 1995), and a SFEB method (Watanabe et al., 2005); InternationalPublication No. 2005/123902). The methods of the present invention cansignificantly improve the survival rate and/or differentiationefficiency of stem cells in a suspension culture.

E. Single Cell Passaging

In some embodiments of pluripotent stem cell culturing, once a culturecontainer is full, the colony is split into aggregated cells or evensingle cells by any method suitable for dissociation, which cell arethen placed into new culture containers for passaging. Cell passaging orsplitting is a technique that enables to keep cells alive and growingunder cultured conditions for extended periods of time. Cells usuallywould be passed when they are about 70%-100% confluent.

Single-cell dissociation of pluripotent stem cells followed by singlecell passaging may be used in the present methods with severaladvantages, like facilitating cell expansion, cell sorting, and definedseeding for differentiation and enabling automatization of cultureprocedures and clonal expansion. For example, progeny cell clonallyderivable from a single cell may be homogenous in genetic structureand/or synchronized in cell cycle, which may increase targeteddifferentiation. Exemplary methods for single cell passaging may be asdescribed in U.S. Pat. App. 20080171385, which is incorporated herein byreference.

In certain embodiments, pluripotent stem cells may be dissociated intosingle individual cells, or a combination of single individual cells andsmall cell clusters comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 cells or more.The dissociation may be achieved by mechanical force, or by a celldissociation agent, such as NaCitrate, or an enzyme, for example,trypsin, trypsin-EDTA, TrypLE Select, or the like.

Based on the source of pluripotent stem cells and the need forexpansion, the dissociated cells may be transferred individually or insmall clusters to new culture containers in a splitting ratio such as atleast or about 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:20, 1:40, 1:50, 1:100,1:150, 1:200, or any range derivable therein. Suspension cell line splitratios may be done on volume of culture cell suspension. The passageinterval may be at least or about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or any range derivabletherein. For example, the achievable split ratios for the differentenzymatic passaging protocols may be 1:2 every 3-7 days, 1:3 every 4-7days, and 1:5 to 1:10 approximately every 7 days, 1:50 to 1:100 every 7days. When high split ratios are used, the passage interval may beextended to at least 12-14 days or any time period without cell loss dueto excessive spontaneous differentiation or cell death.

In certain aspects, single cell passaging may be in the presence of asmall molecule effective for increasing cloning efficiency and cellsurvival, such as a ROCK inhibitor as described above. Such a ROCKinhibitor, e.g., Y-27632, HA-1077, H-1152, or blebbistatin, may be usedat an effective concentration, for example, at least or about 0.02,0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50to about 100 μM, or any range derivable therein.

V. METHODS FOR CARDIAC DIFFERENTIATION

Differentiation of pluripotent stem cells can be induced in a variety ofmanners, such as in attached colonies or by formation of cellaggregates, e.g., in low-attachment environment, wherein thoseaggregates are referred to as embryoid bodies (EBs). The molecular andcellular morphogenic signals and events within EBs mimic many aspects ofthe natural ontogeny of such cells in a developing embryo. However,currently there remain no satisfactory methods for producing largequantities of cardiac cells in a controlled environment. Certain aspectsof the invention disclose methods using non-static suspension culture tocontrol aggregate size and differentiation yield and producecardiomyocytes at a commercial scale.

A. Aggregate Formation

Embryoid bodies (EBs) are aggregates of cells derived from pluripotentstem cells, such as ES cells or iPS cells, and have been studied foryears with mouse embryonic stem cells. In order to recapitulate some ofthe cues inherent to in vivo differentiation, certain aspects of theinvention may employ three-dimensional aggregates (i.e., embryoidbodies) as an intermediate step. Upon aggregation, differentiation isinitiated and the cells begin to a limited extent to recapitulateembryonic development. Though they cannot form trophectodermal tissue(which includes the placenta), cells of virtually every other typepresent in the organism can develop. The present invention may furtherpromote cardiac differentiation following aggregate formation.

Cell aggregation may be imposed by hanging drop, plating upon non-tissueculture treated plates or spinner flasks; either method prevents cellsfrom adhering to a surface to form the typical colony growth. Asdescribed above, ROCK inhibitors may be used before, during or afteraggregate formation to culture pluripotent stem cells.

Pluripotent stem cells may be seeded into aggregate promotion mediumusing any method known in the art of cell culture. For example,pluripotent stem cells may be seeded as a single colony or clonal groupinto aggregate promotion medium, and pluripotent stem cells may also beseeded as essentially individual cells. In some embodiments, pluripotentstem cells are dissociated into essentially individual cells usingmechanical or enzymatic methods known in the art. By way of non-limitingexample, pluripotent stem cells may be exposed to a proteolytic enzymewhich disrupts the connections between cells and the culturing surfaceand between the cells themselves. Enzymes which may be used toindividualize pluripotent stem cells for aggregate formation anddifferentiation may include, but are not limited to, trypsin, in itsvarious commercial formulations, such as TrypIE, or a mixture of enzymessuch as Accutase®.

In certain embodiments, pluripotent cells may be added or seeded asessentially individual (or dispersed) cells to a culturing medium forculture formation on a culture surface. The culturing medium into whichcells are seeded may comprise TeSR medium or mTeSR medium and a survivalfactor such as ROCK inhibitor. For example, dispersed pluripotent cellsare seeded into a culturing medium at a density of from about 10⁴cells/ml to about 10¹⁰ cells/ml. More particularly, pluripotent cellsare seeded at a density of from about 10⁵ cells/ml to about 10⁷cells/ml, or about 0.5×10⁶ cells/ml to about 3×10⁶ cells/ml. In theseembodiments, a culturing surface may be comprised of essentially anymaterial which is compatible with standard aseptic cell culture methodsin the art, for example, a non-adherent surface. A culturing surface mayadditionally comprise a matrix component as described herein. In certainembodiments, a matrix component may be applied to a culturing surfacebefore contacting the surface with cells and medium.

B. Cardiomyocyte Differentiation

Cardiomyocyte lineage cells can be obtained from undifferentiated stemcells by culturing or differentiating in a special growth environmentthat enriches for cells with the desired phenotype (either by outgrowthof the desired cells, or by inhibition or killing of other cell types).

In certain aspects, the iPS cells may be differentiated into cardiaccells in cell suspension incorporating the disclosed methods.Differentiation can be initiated by forming embryoid bodies oraggregates as described above: for example, by overgrowth of apluripotent stem cell culture, or by culturing pluripotent stem cells insuspension in culture vessels having a substrate with low adhesionproperties which allows EB formation. Pluripotent stem cells could beharvested by brief collagenase digestion, dissociated into clusters, andplated in non-adherent cell culture plates (WO 01/51616; U.S. Pat. No.6,602,711, incorporated by reference). Optionally, the EBs can beproduced encapsulated in alginate or other suitable nutrient-permeablematrix, which may help improve the uniformity of EB diameter andconsistency of the cells produced (WO 03/004626, Zandstra et al.,incorporated by reference). Whether or not the process involves EBformation, using a medium that contains serum or serum equivalentpromotes foci of contracting cells of the cardiomyocyte lineage: forexample, about 20% fetal bovine serum, or a serum supplement such as B27or N2 in a suitable growth medium such as RPMI. More exemplary methodsof cardiac differentiation may include embryoid body (EB) methods(Zhang, et al., 2009, which is incorporated by reference), OP9 stromacell methods (Narazaki, et al., 2008, which is incorporated byreference), or growth factor/chemical methods (see U.S. PatentPublication Nos. 20080038820, 20080226558, 20080254003 and 20090047739,all incorporated herein by reference in their entirety).

To promote the cardiomyocyte phenotype, the cells can be cultured withfactors and factor combinations that enhance proliferation or survivalof cardiomyocyte type cells, or inhibit the growth of other cell types.The effect may be due to a direct effect on the cell itself, or due toan effect on another cell type, which in turn enhances cardiomyocyteformation. For example, factors that induce the formation of hypoblastor epiblast equivalent cells, or cause these cells to produce their owncardiac promoting elements, all come within the rubric of cardiotropicfactors or differentiation factors for cardiomyocyte differentiation.

For example, induction medium for cardiac differentiation may include,but is not limited to, precardiac explants, precardiac mesodermconditioned medium, mesoderm secreted growth factors such as HGF.

In certain aspects of the invention, the timing and amount of additionof differentiation factors may be screened for appropriate conditionsfor differentiation of stem cells into cardiomyocytes. In a particularaspect, the differentiation factors may be growth factors that areinvolved in cell development. The differentiation factors may include,but not be limited to, one or more of modulators of signaling pathwaysof bone morphogenetic protein, ActivinA/Nodal, vascular endothelialgrowth factor (VEGF), dickkopf homolog 1 (DKK1), basic fibroblast growthfactor (bFGF), insulin growth factor (IGF), and/or epidermal growthfactor (EGF).

It is contemplated that additional factors may be screened for itsoptimal concentration or timing in the cell differentiation environment,including, but not limited to, fibronectin, laminin, heparin, heparinsulfate, retinoic acid, members of the epidermal growth factor family(EGFs), members of the fibroblast growth factor family (FGFs) includingFGF2, FGF7, FGF8, and/or FGF10, members of the platelet derived growthfactor family (PDGFs), transforming growth factor (TGF)/bonemorphogenetic protein (BMP)/growth and differentiation factor (GDF)factor family agonists or antagonists including but not limited tonoggin, follistatin, chordin, gremlin, cerberus/DAN family proteins, GDFfamily proteins such as GDF-3, ventropin, and amnionless or variants orfunctional fragments thereof. TGF/BMP/GDF antagonists could also beadded in the form of TGF/BMP/GDF receptor-Fc chimeras. Other factorsthat may be screened include molecules that can activate or inactivatesignaling through Notch receptor family, including but not limited toproteins of the Delta-like and Jagged families as well as inhibitors ofNotch processing or cleavage, or variants or functional fragmentsthereof. Other growth factors may include members of the insulin likegrowth factor family (IGE), insulin, the wingless related (WNT) factorfamily, and the hedgehog factor family or variants or functionalfragments thereof. Additional factors may be screened or added topromote mesoderm stem/progenitor, endoderm stem/progenitor, mesodermstem/progenitor, or definitive endoderm stem/progenitor proliferationand survival as well as survival and differentiation of derivatives ofthese progenitors.

Differentiation factors thought to induce differentiation of pluripotentstem cells into cells of the mesoderm layer, or facilitate furtherdifferentiation into cardiomyocyte lineage cells include the followingnon-limiting examples:

Transforming Growth Factor beta-related ligands (exemplified by TGF-β1,TGF-β2, TGF-β3 and other members of the TGF-β3 superfamily illustratedbelow). Ligands bind a TGF-β receptor activate Type I and Type II serinekinases and cause phosphorylation of the Smad effector.

Morphogens like Activin A and Activin B (members of the TGF-βsuperfamily).

Insulin-like growth factors (such as IGF I and IGF II).

Bone morphogenic proteins (members of the TGF-β superfamily, exemplifiedby BMP-2 and BMP-4).

Fibroblast growth factors (exemplified by bFGF, FGF-4, and FGF-8), otherligands that activate cytosolic kinase raf-1 and mitogen-activatedproteins kinase (MAPK), and other mitogens such as epidermal growthfactor (EGF).

Nucleotide analogs that affect DNA methylation and altering expressionof cardiomyocyte-related genes (e.g., 5-aza-deoxy-cytidine).

The pituitary hormone oxytocin, or nitric oxide (NO).

Specific antibodies or synthetic compounds with agonist activity for thesame receptors.

Exemplary effective combinations of cardiotropic agents include use of amorphogen like Activin A and a plurality of growth factors, such asthose included in the TGF-Iβ and IGF families during the earlycommitment stage, optionally supplemented with additional cardiotropinssuch as one or more fibroblast growth factors, bone morphogenicproteins, and platelet-derived growth factors.

Without wishing to be bound by theory, in certain aspects it iscontemplated that TGFβ signaling pathways may be delicately regulated byadjusting timing and level of the external addition of certain growthfactors to achieve optimal specific lineage differentiation condition,such as for differentiation of cardiomyocytes. In certain aspects, theaddition of differentiation factors may help account for variability inthe activity of TGFβ signaling pathway activity for a combination of aselected stem cell clone and a selected culture medium, particularly, inthe relative activity of different TGFβ signaling pathways, such as arelative activity ratio between BMP signaling and Activin signaling.

The transforming growth factor beta (TGFβ) signaling pathway as isinvolved in many cellular processes in both the adult organism and thedeveloping embryo including cell growth, cell differentiation,apoptosis, cellular homeostasis and other cellular functions. In spiteof the wide range of cellular processes that the TGFβ signaling pathwayregulates, the process is relatively simple. TGFβ superfamily ligandsbind to a type II receptor, which recruits and phosphorylates a type Ireceptor. The type I receptor then phosphorylates receptor-regulatedSMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD/coSMADcomplexes accumulate in the nucleus where they act as transcriptionfactors and participate in the regulation of target gene expression.

Stem cells exhibit self-renewing capacity and pluripotency in generatingthe multitude of embryonic and adult cell types of the metazoan body(reviewed by Rossi et al., 2008). Growth factors, such as TGFβ and FGF,regulate stem cell self-renewal and differentiation. FGF2, the mostwidely used growth factor that supports mouse and human embryonic stemcell (ESC) self-renewal in culture, induces TGFβ/activin ligands andreceptors while suppressing BMP-like activities (Greber et al., 2007;Ogawa et al., 2007). Furthermore, pharmacological inhibitors of theTGFβ/nodal type I receptor family suppress human and mouse ESCself-renewal (Ogawa et al., 2007). In general, TGFβ inhibitsdifferentiation of pluripotent progenitor cells, whereas BMP inducestheir differentiation (Watabe and Miyazono, 2009).

To promote self-renewal of ESCs, TGFβ/nodal signaling activates SMAD2and SMAD3, which directly induce Nanog, one of the crucial stem celltranscription factors (Xu, R. H. et al., 2008). TGFβ and FGF signalingsynergize by enhancing binding of Smad complexes to the Nanog promoter.Interestingly, NANOG provides a molecular link for the antagonismbetween TGFβ (the self-renewing factor) and BMP (the differentiationfactor) in ESCs. Nanog binds to SMAD1, inhibiting its transcriptionalactivity and limiting the BMP signaling potential that promotes earlymesodermal differentiation or tissue-specific differentiation later indevelopment (Suzuki et al., 2006). This example is likely to be expandedto additional regulators of ESC self renewal and differentiation as aresult of genome-wide screens for the transcription and signalingfactors of these pathways (Chen et al., 2008).

The TGF Beta superfamily of ligands include: Bone morphogenetic proteins(BMPs), Growth and differentiation factors (GDFs), Anti-mullerianhormone (AMH), Activin, Nodal and TGFβ's. Signaling begins with thebinding of a TGF beta superfamily ligand to a TGF beta type II receptor.The type II receptor is a serine/threonine receptor kinase, whichcatalyses the phosphorylation of the Type I receptor. Each class ofligand binds to a specific type II receptor. In mammals there are sevenknown type I receptors and five type II receptors.

There are three activins: Activin A, Activin B and Activin AB. Activinsare involved in embryogenesis and osteogenesis. They also regulate manyhormones including pituitary, gonadal and hypothalamic hormones as wellas insulin. They are also nerve cell survival factors.

The BMPs bind to the Bone morphogenetic protein receptor type-2 (BMPR2).They are involved in a multitude of cellular functions includingosteogenesis, cell differentiation, anterior/posterior axisspecification, growth, and homeostasis.

The TGF beta family include: TGFβ1, TGFβ2, TGFβ3. Like the BMPS, TGFbetas are involved in embryogenesis and cell differentiation, but theyare also involved in apoptosis, as well as other functions. They bind toTGF-beta receptor type-2 (TGFBR2).

Nodal binds to activin A receptor, type IIB ACVR2B. It can then eitherform a receptor complex with activin A receptor, type IB (ACVR1B) orwith activin A receptor, type IC (ACVR1C).

The TGF beta signaling pathway (Table 24) is involved in a wide range ofcellular process and subsequently is very heavily regulated. There are avariety of mechanisms that the pathway is modulated both positively andnegatively: There are agonists for ligands and R-SMADs; there are decoyreceptors; and R-SMADs and receptors are ubiquitinated.

TABLE 24 TGF beta signaling pathway TGF Beta superfamily Type II Ligandligand Receptor Type I receptor R-SMADs coSMAD inhibitors Activin AACVR2A ACVR1B SMAD2, SMAD4 Follistatin (ALK4) SMAD3 GDF1 ACVR2A ACVR1BSMAD2, SMAD4 (ALK4) SMAD3 GDF11 ACVR2B ACVR1B SMAD2, SMAD4 (ALK4), SMAD3TGFβRI (ALK5) Bone BMPR2 BMPR1A SMAD1 SMAD4 Noggin, morphogenetic(ALK3), SMAD5, Chordin, proteins BMPR1B SMAD8 DAN (ALK6) Nodal ACVR2BACVR1B SMAD2, SMAD4 Lefty (ALK4), SMAD3 ACVR1C (ALK7) TGFβs TGFβRIITGFβRI SMAD2, SMAD4 LTBP1, (ALK5) SMAD3 THBS1, Decorin

In certain embodiments the compositions and methods of the presentinvention comprise adjustment of activity of transforming growth factorbeta (TGF-β) or a TGF-β family member or variants or functionalfragments thereof to determine an appropriate or optimal differentiationcondition.

As used herein, the term “member of the TGF-β family” or the like refersto growth factors that are generally characterized by one of skill inthe art as belonging to the TGF-β family, either due to homology withknown members of the TGF-β family, or due to similarity in function withknown members of the TGF-β family. In particular embodiments of theinvention, if the member of the TGF-β family is present, the TGF-βfamily member of variant or functional fragment thereof activates SMAD 2or 3. In certain embodiments, the member of the TGF-β family is selectedfrom the group consisting of Nodal, Activin A, Activin B, TGF-β, bonemorphogenic protein-2 (BMP2) and bone morphogenic protein-4 (BMP4). Inone embodiment, the member of the TGF-β family is Activin A.

It is contemplated that if Nodal is present, it may be varied from aconcentration of approximately 0.1 ng/mL to approximately 2000 ng/ml,more preferably approximately 1 ng/mL to approximately 1000 ng/ml, morepreferably approximately 10 ng/mL to approximately 750 ng/ml, or morepreferably approximately 25 ng/mL to approximately 500 ng/ml. It iscontemplated that if used, Activin A may be varied at a concentration ofapproximately 0.01 ng/mL to approximately 1000 ng/ml, more preferablyapproximately 0.1 ng/mL to approximately 100 ng/ml, more preferablyapproximately 0.1 ng/mL to approximately 25 ng/ml, or most preferably ata concentration of approximately 6 to 20 ng/ml. It is contemplated thatif present, TGF-β may be varied present at a concentration ofapproximately 0.01 ng/mL to approximately 100 ng/ml, more preferablyapproximately 0.1 ng/mL to approximately 50 ng/ml, or more preferablyapproximately 0.1 ng/mL to approximately 20 ng/ml.

In certain embodiments, the compositions and methods comprise aninhibitor or an inactivator of Activin/Nodal signaling. As used herein,an “inhibitor or inactivator of Activin/Nodal signaling” refers to anagent that antagonizes the activity of one or more Activin/Nodalproteins or any of their upstream or downstream signaling componentsthrough any of its possible signaling pathways. Non-limiting examplesinclude SB-431542.

In certain embodiments, the compositions and methods comprise aninhibitor or an inactivator of BMP signaling. As used herein, an“inhibitor or inactivator of BMP signaling” refers to an agent thatantagonizes the activity of one or more BMP proteins or any of theirupstream or downstream signaling components through any of its possiblesignaling pathways. The compound(s) used to inactivate BMP signaling canbe any compound known in the art, or later discovered. Non-limitingexamples of inhibitors of BMP signaling include dorsomorphin,dominant-negative, truncated BMP receptor, soluble BMP receptors, BMPreceptor-Fc chimeras, noggin, follistatin, chordin, gremlin,cerberus/DAN family proteins, ventropin, high dose activin, andamnionless.

During the elaboration of this invention, it was found that omittingfactors such as insulin-like growth factor II (IGF II) and relatedmolecules from the final stages of in vitro differentiation actuallyincrease the levels of cardiac gene expression. In unrelated studies,IGF II has been found to decrease the levels of GSK3β in fibroblasts(Scalia et al., 2001). IGF II may therefore potentiate the effects ofWnt proteins present in the culture medium or secreted by the cells. Wntproteins normally stabilize and cause nuclear translocation of acytoplasmic molecule, β-catenin, which comprises a portion of thetranscription factor TCF. This changes transcriptional activity ofmultiple genes. In the absence of Wnt, β-catenin is phosphorylated bythe kinase GSK3β, which both destabilizes β-catenin and keeps it in thecytoplasm.

Since Wnt activators like IGF II apparently limit cardiomyocytedifferentiation, certain aspects of this invention may include culturingwith Wnt antagonists to increase the extent or proportion ofcardiomyocyte differentiation of pluripotent stem cells. Wnt signalingcan be inhibited by injection of synthetic mRNA encoding either DKK-1 orCrescent (secreted proteins that bind and inactivate Wnts) (Schneider etal., 2001), or by infection with a retrovirus encoding DKK-1 (Marvin etal., 2001). Alternatively, the Wnt pathway can be inhibited byincreasing the activity of the kinase GSK3β, for example, by culturingthe cells with factors such as IL-6 or glucocorticoids.

In a cerain embodiment, FGF or a combination of FGF and HGF are used toculture pluripotent stem cells, cell aggregates, or differentiated stemcells, which may promote cardiac induction of stem cells. For example,FGF may be added at a concentration of at least or about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150,180, 200, 250 ng/ml or any range derivable therein. Optionally hepaticgrowth factor (HGF) may also be included, for example at a concentrationof at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/ml or any rangederivable therein.

C. Non-static Culture

In certain aspects, non-static culture could be used for cardiacinduction of pluripotent stem cells. Suspension culture can be used toproduce large scale of EBs and cardiomyocytes subsequently; however,static culture has little control over the size and shape of EBs formed,which directly influence yield and quality of cardiomyocytedifferentiated therefrom. The non-static culture can be any culture withcells kept at a controlled moving speed, by using, for example, shaking,rotating, or stirring platforms or culture vessels, particularlylarge-volume rotating bioreactors. The agitation may improve circulationof nutrients and cell waste products and also be used to control cellaggregation by providing a more uniform environment. For example, rotaryspeed may be set to at least or at most about 25, 30, 35, 40, 45, 50,75, 100 rpm, or any range derivable therein. The incubation period inthe non-static culture for pluripotent stem cells, cell aggregates,differentiated stem cells, or cardiomyocytes derivable therefrom, may beat least or about 4 hours, 8 hours, 16 hours, or 1, 2, 3, 4, 5, 6 days,or 1, 2, 3, 4, 5, 6, 7 weeks, or any range derivable therein.

VI. CHARACTERIZATION OF CARDIOMYOCYTE LINEAGE CELLS

The cells obtained according to the techniques of this invention can becharacterized according to a number of phenotypic criteria.Cardiomyocytes and precursor cells derived from pluripotent stem celllines often have morphological characteristics of cardiomyocytes fromother sources. They can be spindle, round, triangular or multi-angularshaped, and they may show striations characteristic of sarcomericstructures detectable by immunostaining. They may form flattened sheetsof cells, or aggregates that stay attached to the substrate or float insuspension, showing typical sarcomeres and atrial granules when examinedby electron microscopy.

Pluripotent stem cell-derived cardiomyocytes and their precursorstypically have at least one of the following cardiomyocyte specificmarkers:

Cardiac troponin I (cTnI), a subunit of troponin complex that provides acalcium-sensitive molecular switch for the regulation of striated musclecontraction.

Cardiac troponin T (cTnT).

Nkx2.5, a cardiac transcription factor expressed in cardiac mesodermduring early mouse embryonic development, which persists in thedeveloping heart.

The cells will also typically express at least one (and often at least3, 5, or more) of the following markers:

Atrial natriuretic factor (ANF), a hormone expressed in developing heartand fetal cardiomyocytes but down-regulated in adults. It is considereda good marker for cardiomyocytes because it is expressed in a highlyspecific manner in cardiac cells but not skeletal myocytes.

myosin heavy chain (MHC), particularly the β chain which is cardiacspecific

Titin, tropomyosin, .alpha.-sarcomeric actinin, and desmin

GATA-4, a transcription factor that is highly expressed in cardiacmesoderm and persists in the developing heart. It regulates many cardiacgenes and plays a role in cardiogenesis

MEF-2A, MEF-2B, MEF-2C, MEF-2D; transcription factors that are expressedin cardiac mesoderm and persist in developing heart

N-cadherin, which mediates adhesion among cardiac cells

Connexin 43, which forms the gap junction between cardiomyocytes.

β1-adrenoceptor (β1-AR)

creatine kinase MB (CK-MB) and myoglobin, which are elevated in serumfollowing myocardial infarction

α-cardiac actin, early growth response-I, and cyclin D2.

Tissue-specific markers can be detected using any suitable immunologicaltechnique—such as flow immunocytometry or affinity adsorption forcell-surface markers, immunocytochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. Antibodies thatdistinguish cardiac markers like cTnI and cTnT from other isoforms areavailable commercially from suppliers like Sigma and SpectralDiagnostics. Expression of an antigen by a cell is said to beantibody-detectable if a significantly detectable amount of antibodywill bind to the antigen in a standard immunocytochemistry or flowcytometry assay, optionally after fixation of the cells, and optionallyusing a labeled secondary antibody.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods using publicly available sequence data (GenBank).Expression of tissue-specific markers as detected at the protein or mRNAlevel is considered positive if the level is at least or about 2-, 3-,4-, 5-, 6-, 7-, 8-, or 9-fold, and more particularly more than 10-, 20-,30, 40-, or 50-fold above that of a control cell, such as anundifferentiated pluripotent stem cell or other unrelated cell type.

Once markers have been identified on the surface of cells of the desiredphenotype, they can be used for immunoselection to further enrich thepopulation by techniques such as immunopanning or antibody-mediatedfluorescence-activated cell sorting.

Under appropriate circumstances, pluripotent stem cell-derivedcardiomyocytes often show spontaneous periodic contractile activity.This means that when they are cultured in a suitable tissue cultureenvironment with an appropriate Ca²⁺ concentration and electrolytebalance, the cells can be observed to contract across one axis of thecell, and then release from contraction, without having to add anyadditional components to the culture medium. The contractions areperiodic, which means that they repeat on a regular or irregular basis,at a frequency between about 6 and 200 contractions per minute, andoften between about 20 and about 90 contractions per minute in normalbuffer. Individual cells may show spontaneous periodic contractileactivity on their own, or they may show spontaneous periodic contractileactivity in concert with neighboring cells in a tissue, cell aggregate,or cultured cell mass.

The contractile activity of the cells can be characterized according tothe influence of culture conditions on the nature and frequency ofcontractions. Compounds that reduce available Ca²⁺ concentration orotherwise interfere with transmembrane transport of Ca²⁺ often affectcontractile activity. For example, the L-type calcium channel blockerdiltiazem inhibits contractile activity in a dose-dependent manner. Onthe other hand, adrenoceptor agonists like isoprenaline andphenylephrine have a positive chronotropic effect. Furthercharacterization of functional properties of the cell can involvecharacterizing channels for Na⁻, K⁺, and Ca⁺. Electrophysiology can bestudied by patch clamp analysis for cardiomyocyte like actionpotentials. See Igelmund et al., 1999; Wobus et al., 1995; andDoevendans et al., 2000.

Functional attributes provide a manner of characterizing cells and theirprecursors in vitro, but may not be necessary for some of the usesreferred to in this disclosure. For example, a mixed cell populationenriched for cells bearing some of the markers listed above, but not allof the functional or electrophysiology properties, can be ofconsiderable therapeutic benefit if they are capable of grafting toimpaired cardiac tissue, and acquiring in vivo the functional propertiesneeded to supplement cardiac function.

Where derived from an established line of pluripotent stem cells, thecell populations and isolated cells of this invention can becharacterized as having the same genome as the line from which they arederived. This means that the chromosomal DNA will be over 90% identicalbetween the pluripotent stem cells and the cardiac cells, which can beinferred if the cardiac cells are obtained from the undifferentiatedline through the course of normal mitotic division. The characteristicthat cardiomyocyte lineage cells are derived from the parent cellpopulation is important in several respects. In particular, theundifferentiated cell population can be used for producing additionalcells with a shared genome—either a further batch of cardiac cells, oranother cell type that may be useful in therapy—such as a populationthat can pre-tolerize the patient to the histocompatibility type of thecardiac allograft (US 2002/0086005; WO 03/050251).

VII. GENETIC ALTERATION OF DIFFERENTIATED CELLS

The cells of this invention can be made to contain one or more geneticalterations by genetic engineering of the cells either before or afterdifferentiation (US 2002/0168766). A cell is said to be “geneticallyaltered” or “transgenic” when a polynucleotide has been transferred intothe cell by any suitable means of artificial manipulation, or where thecell is a progeny of the originally altered cell that has inherited thepolynucleotide. For example, the cells can be processed to increasetheir replication potential by genetically altering the cells to expresstelomerase reverse transcriptase, either before or after they progressto restricted developmental lineage cells or terminally differentiatedcells (US 2003/0022367).

The cells of this invention can also be genetically altered in order toenhance their ability to be involved in tissue regeneration, or todeliver a therapeutic gene to a site of administration. A vector isdesigned using the known encoding sequence for the desired gene,operatively linked to a promoter that is either pan-specific orspecifically active in the differentiated cell type. Of particularinterest are cells that are genetically altered to express one or moregrowth factors of various types such as FGF, cardiotropic factors suchas atrial natriuretic factor, cripto, and cardiac transcriptionregulation factors, such as GATA-4, Nkx2.5, and MEF2-C. Production ofthese factors at the site of administration may facilitate adoption ofthe functional phenotype, enhance the beneficial effect of theadministered cell, or increase proliferation or activity of host cellsneighboring the treatment site.

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector, such as a selectable orscreenable Marker. Such markers would confer an identifiable change tothe cell permitting easy identification of cells containing theexpression vector, or help enrich or identify differentiated cardiaccells by using a cardiac-specific promoter, such as promoters of cardiactroponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavychain (MEW), GATA-4, Nkx2.5, N-cadherin, β1-adrenoceptor, ANF, the MEF-2family of transcription factors, creatine kinase MB (CK-MB), myoglobin,or atrial natriuretic factor (ANF).

Generally, a selectable marker is one that confers a property thatallows for selection. A positive selectable marker is one in which thepresence of the marker allows for its selection, while a negativeselectable marker is one in which its presence prevents its selection.An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, blasticidin, puromycin, hygromycin, DHFR, GPT,zeocin and histidinol are useful selectable markers. In addition tomarkers conferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as chloramphenicol acetyltransferase (CAT) may be utilized.One of skill in the art would also know how to employ immunologicmarkers, possibly in conjunction with FACS analysis. The marker used isnot believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable and screenable markers are well known toone of skill in the art.

VIII. USE OF CARDIOMYOCYTES AND THEIR PRECURSORS

Certain aspects of this invention provide a method to produce largenumbers of cells of the cardiomyocyte lineage. These cell populationscan be used for a number of important research, development, andcommercial purposes.

A. Drug Screening

Cardiomyocytes of this invention can be used commercially to screen forfactors (such as solvents, small molecule drugs, peptides,oligonucleotides) or environmental conditions (such as cultureconditions or manipulation) that affect the characteristics of suchcells and their various progeny.

In some applications, pluripotent stem cells (undifferentiated ordifferentiated) are used to screen factors that promote maturation intolater-stage cardiomyocyte precursors, or terminally differentiatedcells, or to promote proliferation and maintenance of such cells inlong-term culture. For example, candidate maturation factors or growthfactors are tested by adding them to cells in different wells, and thendetermining any phenotypic change that results, according to desirablecriteria for further culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on cardiac muscle tissuemaintenance or repair. Screening may be done either because the compoundis designed to have a pharmacological effect on the cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on cells of this tissue type. The screening can be conductedusing any of the precursor cells or terminally differentiated cells ofthe invention.

The reader is referred generally to the standard textbook In vitroMethods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, either alone or in combinationwith other drugs. The investigator determines any change in themorphology, marker phenotype, or functional activity of the cells thatis attributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to Vickers (pp 375-410 in In vitro Methods in PharmaceuticalResearch, Academic Press, 1997) for further elaboration.

Effect of cell function can be assessed using any standard assay toobserve phenotype or activity of cardiomyocytes, such as markerexpression, receptor binding, contractile activity, orelectrophysiology—either in cell culture or in vivo. Pharmaceuticalcandidates can also be tested for their effect on contractileactivity—such as whether they increase or decrease the extent orfrequency of contraction. Where an effect is observed, the concentrationof the compound can be titrated to determine the median effective dose(ED₅₀).

B. Animal Testing

Certain aspects of this invention also provide for the use ofcardiomyocytes and their precursors to enhance tissue maintenance orrepair of cardiac muscle for any perceived need, such as an inborn errorin metabolic function, the effect of a disease condition, or the resultof significant trauma.

To determine the suitability of cell compositions for therapeuticadministration, the cells can first be tested in a suitable animalmodel. At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Cell compositions are administered toimmunodeficient animals (such as nude mice, or animals renderedimmunodeficient chemically or by irradiation). Tissues are harvestedafter a period of regrowth, and assessed as to whether pluripotent stemcell-derived cells are still present.

This can be performed by administering cells that express a detectablelabel (such as green fluorescent protein, or β-galactosidase); that havebeen prelabeled (for example, with BrdU or [³H]thymidine), or bysubsequent detection of a constitutive cell marker (for example, usinghuman-specific antibody). The presence and phenotype of the administeredcells can be assessed by immunohistochemistry or ELISA usinghuman-specific antibody, or by RT-PCR analysis using primers andhybridization conditions that cause amplification to be specific forhuman polynucleotides, according to published sequence data.

Suitability can also be determined by assessing the degree of cardiacrecuperation that ensues from treatment with a cell population ofcardiomyocytes derived from pluripotent stem cells. A number of animalmodels are available for such testing. For example, hearts can becryoinjured by placing a precooled aluminum rod in contact with thesurface of the anterior left ventricle wall (Murry et al., 1996;Reinecke et al., 1999; U.S. Pat. No. 6,099,832; Reinecke et al., 2004).In larger animals, cryoinjury can be effected by placing a 30-50 mmcopper disk probe cooled in liquid N2 on the anterior wall of the leftventricle for about 20 min (Chiu et al., 1995). Infarction can beinduced by ligating the left main coronary artery (Li et al., 1997).Injured sites are treated with cell preparations of this invention, andthe heart tissue is examined by histology for the presence of the cellsin the damaged area. Cardiac function can be monitored by determiningsuch parameters as left ventricular end-diastolic pressure, developedpressure, rate of pressure rise, and rate of pressure decay.

C. Therapeutic Use in Humans

After adequate testing, differentiated cells of this invention can beused for tissue reconstitution or regeneration in a human patient orother subject in need of such treatment. The cells are administered in amanner that permits them to graft or migrate to the intended tissue siteand reconstitute or regenerate the functionally deficient area. Specialdevices are available that are adapted for administering cells capableof reconstituting cardiac function directly to the chambers of theheart, the pericardium, or the interior of the cardiac muscle at thedesired location.

Where desirable, the patient receiving an allograft of pluripotent stemcell-derived cardiomyocytes can be treated to reduce immune rejection ofthe transplanted cells. Methods contemplated include the administrationof traditional immunosuppressive drugs like cyclosporin A (Dunn et al.,Drugs 61:1957, 2001), or inducing immunotolerance using a matchedpopulation of pluripotent stem cell-derived cells (WO 02/44343; U.S.Pat. No. 6,280,718; WO 03/050251). Another approach is to adapt thecardiomyocyte cell population to decrease the amount of uric acidproduced by the cells upon transplantation into a subject, for example,by treating them with allopurinol. Alternatively or in conjunction, thepatient is prepared by administering allopurinol, or an enzyme thatmetabolizes uric acid, such as urate oxidase (PCT/US04/42917).

Patients suitable for receiving regenerative medicine according to thisinvention include those having acute and chronic heart conditions ofvarious kinds, such as coronary heart disease, cardiomyopathy,endocarditis, congenital cardiovascular defects, and congestive heartfailure. Efficacy of treatment can be monitored by clinically acceptedcriteria, such as reduction in area occupied by scar tissue orrevascularization of scar tissue, and in the frequency and severity ofangina; or an improvement in developed pressure, systolic pressure, enddiastolic pressure, patient mobility, and quality of life.

The cardiomyocytes of this invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. Incertain aspects, it may be desirable to disperse the cells using aprotease or by gentle mechanical manipulation into a suspension ofsingle cells or smaller clusters. To reduce the risk of cell death uponengraftment, the cells may be treated by heat shock or cultured withabout 0.5 U/mL erythropoietin about 24 hours before administration.

For general principles in medicinal formulation, the reader is referredto Cell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, 1996; and Hematopoetic Stem Cell Therapy, 2000. Choice ofthe cellular excipient and any accompanying elements of the compositionwill be adapted in accordance with the route and device used foradministration. The composition may also comprise or be accompanied withone or more other ingredients that facilitate the engraftment orfunctional mobilization of the cardiomyocytes. Suitable ingredientsinclude matrix proteins that support or promote adhesion of thecardiomyocytes, or complementary cell types, especially endothelialcells.

This invention also includes a reagent system, comprising a set orcombination of cells that exist at any time during manufacture,distribution, or use. The cell sets comprise any combination of two ormore cell populations described in this disclosure, exemplified but notlimited to a type of differentiated cell (cardiomyocytes, cardiomyocyteprecursors, and so on), in combination with undifferentiated pluripotentstem cells or other differentiated cell types, often sharing the samegenome. Each cell type in the set may be packaged together, or inseparate containers in the same facility, or at different locations, atthe same or different times, under control of the same entity ordifferent entities sharing a business relationship.

Pharmaceutical compositions of this invention may optionally be packagedin a suitable container with written instructions for a desired purpose,such as the reconstitution of cardiomyocyte cell function to improve adisease condition or abnormality of the cardiac muscle.

IX. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Single Cell Splitting for Automation

IPS cells were maintained on Matrigel™ in TeSR medium (from Stem CellTechnologies) in 6-well plates. After the media were aspirated, 1ml/well of 0.05% Trypsin was added and the cells were incubated at 37°C. for 7 minutes. Equal volume of A1 media (TeSR media containing 1 μMH-1152 (or HA-100) (EMD), 0.5 mg/ml Soybean Trypsin inhibitor(Invitrogen)) were added to the cells. Then the cells were seeded intoMatrigel™-coated vessels containing the A1 media and fed every 24 hourswith TeSR media.

Example 2 Single Human IPS Cell Passaging with NaCitrate

Dissociated iPS cells were rinsed with D-PBS (Dulbecco's PhosphateBuffered Saline) free of Ca²⁺ and Mg²⁺ and the media were aspirated. Thecells were then incubated with appropriate amount (i.e., 1 mL for onewell of 6-well plate, 15 mL for T-150 flask, etc) of 15 mM NaCitrate,165 mM KCl, pH 7.3, OSM 340, at room temperature for 6-15 minutes. Afterincubation, the NaCitrate solution were aspirated with the cells stillloosely attached. The cells were resuspended in appropriate amount ofTeSR with 1 μM H1152 and distributed to Matrigel™ surfaced vessel.

For scale up production of the starting material from T150 flasks toCorning Double Stacks, and for assessment of the ability of iPS cells tobe passaged as single cells/small clumps with NaCitrate, iPS 6.1 and iPS6.1 MRB were passaged with 15 mM NaCitrate as single cells or small cellclumps into double stacks while maintaining T150 control flasks splitwith dispase (see FIG. 1 for experimental design). The Double StackNaCitrate conditions had a good typical doubling time of ˜30 hrs (FIGS.2A-C). Cells were assessed at passage 4 and passage 7 withcardiogenesis, pluripotency panel, and karyotype. Cells were split 4times with NaCitrate and maintained a normal karyotype (FIG. 3).Aggregate images and cTnT staining for cardiomyocytes were analyzed onday 14 after splitting (FIGS. 4A-D, FIGS. 5A-C, FIGS. 6A-B, FIGS. 7A-C).Pluripotency profiles of iPS cells after passaging with NaCitrate anddispase was compared by flow cytometry using five pluripotency markers(FIGS. 8A-C). It appears safe to passage iPS cells at least four timeswith the NaCitrate method.

Cardiogenesis was also analyzed for those iPS cells after passaging withNaCitrate and data were compiled in FIG. 9. The figure shows thecardiomyocytes generated from iPS6.1 and iPS6.1 MRB cells passagednormally with dispase in T150 flasks (control) as well as cells passagedin Double stacks with NaCitrate. The last three “spinner” samples comefrom Double Stack iPS 6.1 MRB cells that were left over from variousDouble stack harvests. An average yield of 110e6 cells/L was obtained in1L spinner with cells passaged in double stacks with NaCitrate.

The experiments and data described above support the use of NaCitrate asa means to split iPS cells such as iPS 6.1 and iPS 6.1 MRB. Combiningthis split method with Double Stack flasks will allow more simple andefficient production of large quantities of starting material. NaCitratesplit cells spread out much more evenly and the cells can be grown tomore of a monolayer than cells split with dispase. This allows theinventors to maximize both the surface area and TeSR usage/cell for eachflask. NaCitrate also solves the problem of needing top access to thecells in order to scrape them off when using dispase. It appears theinventors can safely passage iPS cells at least 4 times with NaCitrate(from dispase maintained cells) and still have the cells which maintainnormal karyotype, cardiogenic potential, and pluripotency.

Example 3 Induction of IPS cells into Cardiomyocytes via SuspensionAggregates in a 1L Spinner Flask

Human pluripotent stem cells were maintained and expanded on Matrigel™in TeSR medium into cardiomyocytes via suspension aggregates in a 1Lspinner flask as in the following experiments.

IPS-MRB cells were maintained on Matrigel™ in TeSR medium (from StemCell Technologies) no later than when they would normally be ready to bepassaged. IPS cells were grown in T-150 flasks throughout this example,but can be scaled for starting cells in other culture formats. FiveT-150 flasks of iPS cells were harvested: media were aspirated and cellswere incubated with 12 mL room temperature TrypLe™ (Invitrogen), arecombinant typsin-like enzyme, at 37° C. for 7 minutes. The cells werethen transferred to five 50 ml conical tubes (one tube for each T-150flask) containing 12 ml DMEM/F12 (GIBCO) with 10% fetal calf serum (FCS)(Chemicon). The cells were then pelleted at 1200 rpm for 5 minutes andsupernatant aspirated. The pellet was resuspended in 10 mL aggregatepromotion medium (TeSR medium containing 25 μg/ml Gentamicin, 1 μMH1152, and with or without 50 ng/ml human recombinant HGF). Theresuspended pellet were combined into a single 250 ml conical tube(allowing volume for later dilution), if necessary. Cells were countedusing CEDEX HiRES cell counter and then diluted to 1.0×10⁶ cells/mL.Each 500 mL diluted cell stock was dispensed into one 1 L spinner flask(leaving room in spinner flasks for later dilution) and the flasks wereplaced on magnetic stir platform with a speed of 50 RPM (both side capswere loosed by two full turns to allow gas transfer).

On the next day (Day 1), flasks containing the cells were set in cellculture hood and suspended aggregate allowed to settle to the bottom ofthe flasks for 30 minutes. The spent media were aspirated using a 2 mLaspirating pipet with approximately 100 mL left in the flasks (if filmsof cells form on bottom after setting, shake flask back and forth ineach direction to break them up before adding medium). Aggregatetransition medium (50% TeSR medium, 45% DMEM containing GlutaMax™, 5%ES-qualified FCS, 25 μg/ml Gentamicin, 1 μM H1152, and with or without50 ng/ml HGF) were added to the flasks using Pipeline machine to bringto the original volume. The flasks were returned to the stirringplatform inside a 7% CO₂ incubator and the agitator was started tooperate at 50±1 RPM (both side caps were loosed by two full turns toallow gas transfer).

On Day 2, flasks containing the cells were set in cell culture hood andsuspended aggregate allowed to settle to the bottom of the flasks for 15minutes. The spent media were aspirated with approximately 100 mL leftin the flasks. Aggregate suspension were gently mixed in flasks, andcardiac induction medium (90% TeSR medim, 10% ES-qualified FCS, 25 μg/mlGentamicin, 50 ng/ml human recombinant bFGF, and with or without 50ng/ml human recombinant HGF) were added to each flask to 1 L (2×dilution) with Pipeline machine. The flasks were returned to thestirring platform inside a 7% CO₂ incubator and the agitator was startedto operate at 50±1 RPM (both side caps were loosed by two full turns toallow gas transfer).

On Day 3, flasks containing the cells were set in cell culture hood andsuspended aggregate allowed to settle to the bottom of the flasks for 15minutes. The spent media were aspirated with approximately 100 mL leftin the flasks. Spinner flasks were shaken back and forth to break largeclumps. Cardiac induction medium were added back to each flask to bringback to the original volume for Days 3-7; cardiac maintenance mediumwere exchanged instead for Days 8, 10, 12. After every media exchange,the flasks were returned to the stirring platform inside a 7% CO₂incubator and the agitator was started to operate at 50±1 RPM (both sidecaps were loosed by two full turns to allow gas transfer).

Cell cultures were analyzed on Day 14 by total cell counting per flaskat harvest, cTNT or RFP purity at harvest. Aggregate count was alsocarried on Day 2 and final stage and aggregate pictures were taken.

EXAMPLE 4 Induction of IPS Cells into Cardiomyocytes via SuspensionAggregates in T-Flasks

IPS or hES cells (either iPS6.1 cells, iPS6.1-MRB cells, or H9-TGZcells) were maintained on Matrigel™ in TeSR medium (from Stem CellTechnologies) no later than when they would normally be ready to bepassaged. The starting cells were grown in T-150 flasks, but could bescaled in other culture formats.

For aggregate formation on the first day (Day 0), ultra low attachment(ULA) T75 or T25 flasks by marking feeding line were prepared. A feedingline marking template was placed against the raised edge on the top sideof a low attachment flask (FIG. 10A) and a line was drawn on the flaskwith a permanent (alcohol resistant) marker (FIG. 10B). The flask werelabeled then.

Up to five T150 flasks of cells were harvested at a time: cells wereexamined and verified to be 60-80% confluent. Media were aspirated and12 mL room temperature TrypLE were added to incubate at 37° C. for 7minutes. Meanwhile, one 50 mL conical tube was prepared for each T150 byadding 12 mL DMEM/F12 with 10% FCS. After 7 minute incubation, theflasks were tapped to dislodge residual cells and cells were pipettedinto prepared 50 mL tubes. Cells were then pelleted at 1200 rpm, 5minutes using centrifuge and supernatant was aspirated withoutdisturbing the cell pellet. The cell pellet were resuspended in 10 mLaggregate formation medium as described in Example 3.

Multiple tubes of cells were combined into a single 250 mL conical tube(allowing volume for later dilution), if necessary. Cells were countedusing CEDEX HiRES cell counter according to SOP EQ-15 and then dilutedto 1.0×10⁶ cells per mL. Each 15 mL diluted cell stock was dispensedinto each ULA T75 (or 5 mL per ULA T25) flask.

The flasks/plates were placed on rocker platform inside 7% CO₂ incubatorto incubate for 24±4 hrs. Be sure rocker is well-balanced. The rockerwas started with the agitator operating at 7.5±2 RPM for T75 flasks or15±1 RPM for T25 flasks. To measure rocker RPM, a clock or timer wasused to keep time, and the number of complete cycles was counted in 60seconds. The rotary speed was adjusted and re-measured until RPM readingfalls within specifications. Re-measurement and readjustment wereperformed as necessary anytime flasks were added or removed.

The next day (Day 1) the cells were fed aggregate transition media:cells were fed by tilting flask on short edge at a 45° angle as shown inFIG. 11A and suspended aggregates were allowed to settle to the bottomcorner of the flask for 10 minutes. Spent media were aspirated with aPasteur pipet down to the feeding line drawn on the flask (FIG. 11B).Care should be taken to prevent scratching the low attachment surface ofthe flask with the pipet as aggregates could stick to the damagedsurface. The flask was turned upright and 15 mL/T75 (or 5 mL/T25) flaskwas gently added the Aggregate Transition Medium.

The flasks/plates were placed on rocker platform inside 7% CO₂ incubatorto incubate for 24±4 hrs. The rocker was started with the agitatoroperating at 7.5±2 RPM for T75 flasks or 15±1 RPM for T25 flasks. Rotaryspeed was adjusted as described above.

On Day 2, cells were diluted and distributed to T25 or T75 flasks.First, additional ULA T25 or T75 flasks were prepared by marking feedingline as described above and labeling. Cells were fed by tilting flask onshort edge at a 45° angle as shown in FIG. 11A and suspended aggregateswere allowed to settle to the bottom corner of the flask for 10 minutes.Spent media were aspirated with a Pasteur pipet down to the feeding linedrawn on the flask (FIG. 11B). The flask was turned upright and 15mL/T75 (or 5 mL/T25) flask was gently added appropriate volume ofcardiac induction medium (30 ml for T75 flask or 10 ml for T25 flask).If large clumps of cells have formed, the resuspended aggregates may befiltered through a 200 um sterile mesh filter into a 50 mL conical tubebefore proceeding.

Then the cells were distributed to appropriate sized flasks. If seedingto T25 flasks: 5 mL of thoroughly resuspended aggregates weretransferred to each of six T25 flasks. The goal is to seed theaggregates formed from 2.5 million cells into each T25 (i.e., 0.5million cells per mL). Do not correct for cell survival or aggregatenumber. If seeding to T75 flasks: 15 mL of thoroughly resuspendedaggregates were transferred to each of two T75 flasks. The goal is toseed the aggregates formed from 7.5 million cells into each T75 (i.e.,0.5 million cells per mL). Do not correct for cell survival or aggregatenumber.

The flasks/plates were placed on rocker platform inside 7% CO₂ incubatorto incubate for 24±4 hrs. The rocker was started with the agitatoroperating at 7.5±2 RPM for T75 flasks or 15±1 RPM for T25 flasks. Rotaryspeed was adjusted as described above.

On Day 3-13, cells were by tilting flask on short edge at a 45° angle asshown in FIG. 11A and allow suspended aggregates to settle to the bottomcorner of the flask for 10 minutes. Spent media were aspirated with aPasteur pipet down to the feeding line drawn on the flask (FIG. 11B).

The flask was turned upright and gently added the media (5 mL media perT25 flask or 15 mL media per T75 flask) described below: Days 3, 4, 5,6, 7: Cardiac Induction Medium; Days 8, 10, 12: Cardiac MaintenanceMedium

The flasks/plates were placed on rocker platform inside 7% CO₂ incubatorto incubate for 24±4 hrs. The rocker was started with the agitatoroperating at 7.5±2 RPM for T75 flasks or 15±1 RPM for T25 flasks. Rotaryspeed was adjusted as described above.

Cultures of cells were be terminated and analyzed on day 14: total cellcount per flask at harvest, cTNT purity (or GFP purity) were analyzed atharvest. Aggregates were counted and imaged on Day 2 and at harvest.

EXAMPLE 5 Aggregate Formation

IPS cells were treated with dispase and then incubated with TrypLe™(Invitrogen) to dissociate cells. Dissociated iPS cells were split andpassaged by 1:6 dilution. The TrypLe™ split and passaging was repeatedonce. Four densities of iPS cells were seeded for aggregate formation:0.5×10⁶, 1×10⁶, 2×10⁶, and 3×10⁶ cells/ml (FIG. 12). Aggregate formationand cardiac induction procedures of those iPS cells at different initialdensities were performed essentially the same as described in Examples4. Step yield (i.e., percentage of cells incorporated into aggregates),aggregate size, yield of cardiac cells are measured and detected inFIGS. 13-17. Aggregate formation at different rotary speed were alsocompared as in Table 2 below.

TABLE 2 Aggregate Formation and Rotary Speed Spinner #1 Spinner #2P-Value Spinner speed on day 0 50 rpm 70 rpm # of samples (with areabetween 171 151 50 μm² and 1000 μm²) Average Area (μm²) 235.32 137.44371STD AREA (μm²) 124.55897 94.20054 2.36234E−14

Previous experiments by the inventors that used ROCK inhibitors andtrypsin to form aggregates from H1 and IPS cells combined with singlecell splitting techniques, showed proof of principle that it is possibleto form beating cardiomyocytes in a suspension culture using singlecells. This information led to the designation of experiments with thegoal to develop an aggregate formation method with high throughput andhigh step-yield (i.e. percentage of cells incorporated into aggregates)using iPS cells. The purpose of the following experiments is to showthat the ROCK inhibitor H₁₁₅₂ allows IPS cells, individualized withTrypLE, to self aggregate, and to form cardiomyocytes in cardiacdifferentiation media.

Critical reagents were obtained from the following sources: DMEM andGlutamax were from Gibco (#10567); fetal calf serum was from Chemicon(#ES-009B); human recombinant HGF (hrHGF) and human recombinant basicFGF were from the R&D systems (234-FSE and 294-HGN, respectively).Gentamicin was from Gibco (#15750). H₁₁₅₂ was from Calbiochem (#55550).

The first experiment (Experiment 1, see FIG. 18) described in thisExample showed the potential of the single cell aggregate formationmethod for cardiomyocyte differentiation. The second experiment(Experiment 2, see FIG. 19) showed the potential of single cellaggregates using cells maintained with dispase. Cardiomyocytedifferentiation conditions are summarized in Table 3-4 and 5-6 forExperiment 1 and 2, respectively.

TABLE 3 Summary of Cardiomyocyte differentiation conditions inExperiment 1 Cell Number Dissociation per Conditions Reagent VesselVessel notes Static TrypLE trypsin 18.5e6 T75 Static, NAB cultured,assayed day 15 Dynamic TrypLE trypsin 18.5e6 T75 Placed on rotator after2 hours, CS cultured cells after day 0, assayed day 15

TABLE 4 Experimental Parameters form Experiment 1 Parameter MethodTimepoint Cell morphology Phase contrast Day1, day 3, and day 15 atmicroscopy end of cardio process Cell Counts Cell count by Cedex End ofthe cardio process Cardiomyocyte Flow cytometry End of the cardioprocess differentiation

TABLE 5 Summary of Cardiomyocyte differentiation conditions inExperiment 2 Cell Dissociation Number Conditions Reagent per VesselVessel notes 3e5 cells/ml TrypLE trypsin 1.5e6 T25 One day Static, thenagitated 5e5 cells/ml TrypLE trypsin 2.5e6 T25 One day Static, thenagitated 7e5 cells/ml TrypLE trypsin 3.75e6  T25 One day Static, thenagitated 10e5 cells/ml  TrypLE trypsin   5e6 T25 One day Static, thenagitated

TABLE 6 Experimental Parameters form Experiment 2 Parameter MethodTimepoint Cell Phase contrast microscopy Day 8, also aggregatemorphology counts before each feeding. Cell Counts Cell count by CedexEnd of the cardio process Cardiomyocyte Flow cytometry End of the cardioprocess differentiation

Cell morphology and aggregate pictures from Experiment 1 and 2 wereshown in FIGS. 20-21, respectively. After 1 day in aggregate formationmedia aggregation of the IPS cells was clearly visible, these aggregatestended to be more uniform in size then aggregates form using the dispasemethod. The data presented in FIGS. 20-21 show consistent aggregateformation across different seeding densities and between cellsmaintained as single cells and those with dispase.

Cells were also counted for both experiments (Tables 7-8) and the cellcount represented number of cell per flask on day 15 at the end of thedifferentiation process.

TABLE 7 Cell/aggregate counts from Experiment 1 Total Aggregates/ Cells/cells/T75 T75 flask Aggregate Flask Agitated  739 ± 155 3112 2.3 * 10⁶Static 4140 ± 945 1787 7.4 * 10⁶

TABLE 8 Average cell counts from Experiment 2 Average Cell Count perflask (in millions) T25_3e5 0.112 ± 0.129 T25_5e5 0.343 ± 0.161 T25_7e50.587 ± 0.333 T25_10e5 0.951 ± 0.229

Cardiomyocyte differentiation was also analyzed by using Troponin Tstaining (Tables 9-10). Troponin T results represented the number ofcardiomyocytes per flask on day 15 at the end of the differentiationprocess.

TABLE 9 Cardiomyocyte differentiation from Experiment 1 Percent PercentPercent Percent troponin T+ Troponin T+ MF20+ MF20+ Day 15 Day 15 Day 15Day 15 (replicate 1) (replicate 2) (replicate 1) (replicate 2) Static1.07 0.94 1.02 0.96 Agitated 9.01 8.77 6.99 7.36

TABLE 10 Cardiomyocyte differentiation from Experiment 2 Average AverageCell Count per flask Average % Average ratio iPS to Average # cTnT (inmillions) troponin T + Cardiomyocyte Yield CM Cells/Liter* T25_3e5 0.112± 0.129 11.66 ± 1.86 11726 ± 12908 491 ± 639 1.95e6 ± 2.15e6 T25_5e50.343 ± 0.161 11.87 ± 1.60 40227 ± 17737 75 ± 44 6.70e6 ± 2.95e6 T25_7e50.587 ± 0.333  7.07 ± 0.74 40200 ± 19175 98 ± 47 6.69e6 ± 3.19e6T25_10e5 0.951 ± 0.229  5.53 ± 0.33 52723 ± 14268 99 ± 25 8.78e6 ±2.37e6

The experiments shown in this Example demonstrated the potential ofsingle cell aggregate formation in the cardiomyocytes differentiation.Using single cells allows for easy quantification of the number of cellput into the differentiation system allowing for further optimization ofcell seeding density, and more control over the differentiation process.The data from the experiments show that the cardiomyocytes yield isequal to or greater than data from the dispase method. The use of H1152and TrypLE allows for a scalable high throughput method of aggregateformation.

EXAMPLE 6 H₁₁₅₂ Duration and Aggregate Formation

The compound H₁₁₅₂ has been shown to allow individualized pluripotentcells to self aggregate. The purpose of this Example was to see theeffect of 1 day or 2 day treatment with the compound on Cardiomyocytedevelopment. Initial testing with H₁₁₅₂ included the compound in themedia: 1 μM on day one with the TeSR media and 0.5 μM on day 2 with thecompound in the aggregate transition media. After adopting a new methodwhich included the H₁₁₅₂ compound for 1 day at 1 μM only, there appearedto be a loss of efficiency with the initial cell survival, resulting inthis study to determine if extended H₁₁₅₂ would increase cell survivalwithout a negative impact on carcinogenesis. Two experiments weredesigned as shown in FIGS. 22-23. Cardiomyocyte differentiationconditions and experimental parameters were summarized in Tables 11-12and 13-14 for Experiments 1 and 2, respectively. Critical reagents andmaterials were obtained as described in the previous example.

Final results from Experiment 1 (Table 15)after 14 days in cardiomyocytedifferentiation. Increasing H₁₁₅₂ exposure duration to 48 hoursincreased cTnT yield in each cell density condition. Final results fromExperiment 2 (Table 16) after 14 days in cardiomyocyte differentiation.Increasing H₁₁₅₂ exposure duration to 48 hours increased cTnT yield inthe cultures initially seeded with 3e5 and 5e5 cells/mL. Results ofaggregate formation and cardiac differentiation for the experiments werepresented in FIGS. 24A-J and FIGS. 25-27.

Two day treatment of H1152 at 1 μM had a significant increase in thecell survival in each of the experiments. There was a difference in thepurity of cTnT positive cells between one and two day treatments whichcorrelates with the cell survival and thus the cell density. Theinventors contemplated that the decrease in purity with extended H1152treatment is due to increased overall cell density, though thepossibility of a direct effect of H1152 on cardiogenesis remains. Theincrease in cell survival due to the two day treatment of the compoundis more significant than the decrease in cTnT purity resulting in largeryields of cTnT positive cells.

Effect of different concentrations of H1152 was also tested on cellsurvival with one day dose of H1152 on IPS 6.1 cells (FIG. 28). Thisdata suggested that 1 μM concentration was the optimal concentration foraggregate formation.

TABLE 11 Summary of Cardiomyocyte differentiation conditions inExperiment 1 Cell Number Dissociation per Conditions Reagent VesselVessel notes 3e5 cells/ml TrypLE trypsin 1.5e6 T25 H₁₁₅₂ in Cardiacformation media only, 24 hour exposure @ 1 μM 5e5 cells/ml TrypLEtrypsin 2.5e6 T25 H₁₁₅₂ in Cardiac formation media only, 24 hourexposure @ 1 μM 7e5 cells/ml TrypLE trypsin 3.75e6  T25 H₁₁₅₂ in Cardiacformation media only, 24 hour exposure @ 1 μM 10e5 cells/ml  TrypLEtrypsin   5e6 T25 H₁₁₅₂ in Cardiac formation media only, 24 hourexposure @ 1 μM 15e5 cells/ml  TrypLE trypsin 7.5e6 T25 H₁₁₅₂ in Cardiacformation media only, 24 hour exposure @ 1 μM 3e5 cells/ml TrypLEtrypsin 1.5e6 T25 H₁₁₅₂ in Cardiac formation media and transition media,48 hour exposure @ 1 μM 5e5 cells/ml TrypLE trypsin 2.5e6 T25 H₁₁₅₂ inCardiac formation media and transition media, 48 hour exposure @ 1 μM7e5 cells/ml TrypLE trypsin 3.75e6  T25 H₁₁₅₂ in Cardiac formation mediaand transition media, 48 hour exposure @ 1 μM 10e5 cells/ml  TrypLEtrypsin   5e6 T25 H₁₁₅₂ in Cardiac formation media and transition media,48 hour exposure @ 1 μM 15e5 cells/ml  TrypLE trypsin 7.5e6 T25 H₁₁₅₂ inCardiac formation media and transition media, 48 hour exposure @ 1 μM

TABLE 12 Experimental Parameters for Experiment 1 Parameter Method Timepoint Cell morphology Phase contrast microscopy Day 14 Cell Counts Cellcount by Cedex End of the cardio process Cardiomyocyte Flow cytometryEnd of the cardio process differentiation

TABLE 13 Summary of Cardiomyocyte differentiation conditions inExperiment 2 Cell Number Dissociation per Conditions Reagent VesselVessel notes  3e5 cells/ml TrypLE trypsin 1.5e6 T25 H₁₁₅₂ in Cardiacformation media only, 24 hour exposure @ 1 μM  5e5 cells/ml TrypLEtrypsin 2.5e6 T25 H₁₁₅₂ in Cardiac formation media only, 24 hourexposure @ 1 μM 10e5 cells/ml TrypLE trypsin   5e6 T25 H₁₁₅₂ in Cardiacformation media only, 24 hour exposure @ 1 μM 15e5 cells/ml TrypLEtrypsin 7.5e6 T25 H₁₁₅₂ in Cardiac formation media only, 24 hourexposure @ 1 μM  3e5 cells/ml TrypLE trypsin 1.5e6 T25 H₁₁₅₂ in Cardiacformation media and transition media, 48 hour exposure @ 1 μM  5e5cells/ml TrypLE trypsin 2.5e6 T25 H₁₁₅₂ in Cardiac formation media andtransition media, 48 hour exposure @ 1 μM 10e5 cells/ml TrypLE trypsin  5e6 T25 H₁₁₅₂ in Cardiac formation media and transition media, 48 hourexposure @ 1 μM 15e5 cells/ml TrypLE trypsin 7.5e6 T25 H₁₁₅₂ in Cardiacformation media and transition media, 48 hour exposure @ 1 μM

TABLE 14 Experimental Parameters for Experiment 2 Parameter Method Timepoint Cell Counts Cell count by Cedex End of the cardio processCardiomyocyte Flow cytometry End of the cardio process differentiation

TABLE 15 Summary of Results for Experiment 1 Average Cell Average #Count per Average Average cTnT flask Average % Cardiomyocytes ratio iPSCells/Liter (in Density 3.0 (in millions) Troponin T + Yield to CMmillions) 3e5_24 Hours 0.25 ± 0.11 6.83 ± 0.26 17445 ± 7677  99 ± 452.91 ± 1.27 3e5_48 Hours 1.39 ± 0.28 4.40 ± 1.00 59575 ± 1514  25 ± 1 9.92 ± 0.25 5e5_24 Hours 0.46 ± 0.48 6.26 ± 3.40 18123 ± 10004 180 ± 1193.02 ± 1.66 5e5_48 Hours 3.41 ± 0.47 3.37 ± 1.12 117137 ± 48094  24 ± 1119.52 ± 8.01  7e5_24 Hours 0.77 ± 0.29 5.41 ± 1.58 39272 ± 5494  90 ± 126.54 ± 0.91 7e5_48 Hours 3.19 ± 1.81 2.23 ± 0.28 70299 ± 40479 72 ± 5811.71 ± 6.74  10e5_24 Hours  1.95 ± 0.91 3.29 ± 2.73 51174 ± 22025 116 ±66  8.53 ± 3.67 10e5_48 Hours  4.69 ± 0.64 4.70 ± 1.08 224015 ± 71410 24 ± 9  37.33 ± 11.90 15e5_24 Hours  4.01 ± 0.46 2.05 ± 0.89 80131 ±31034 105 ± 43  13.35 ± 5.17  15e5_48 Hours  6.16 ± 1.31 4.84 ± 1.61299246 ± 127411 28 ± 10 49.87 ± 21.23

TABLE 16 Summary of Results for Experiment 1 Average Cell Average #Count per Average Average cTnT flask Average % Cardiomyocytes ratio iPSCells/Liter (in Density 3.1 (in millions) Troponin T + Yield to CMmillions)  3e5_24 Hours 0.57 ± 0.54 12.71 ± 1.72  66383 ± 54471 33 ± 2011.06 ± 9.07   3e5_48 Hours 2.52 ± 0.51 7.99 ± 2.24 208066 ± 94874  8 ±4 34.67 ± 15.81  5e5_24 Hours 0.52 ± 0.35 9.98 ± 2.40 53055 ± 36109 67 ±49 8.84 ± 6.01  5e5_48 Hours 4.56 ± 0.48 5.20 ± 3.33 230454 ± 138874 14± 7  38.40 ± 23.14 10e5_24 Hours 1.54 ± 063  5.42 ± 1.08 85129 ± 4409272 ± 42 14.18 ± 7.34  10e5_48 Hours 7.84 ± 1.14 1.40 ± 1.45 96206 ±81824 77 ± 44 16.03 ± 13.63 15e5_24 Hours 2.63 ± 0.14 6.48 ± 2.42 171264± 65034  48 ± 17 28.54 ± 10.83 15e5_48 Hours 6.16 ± 1.31 2.36 ± 2.03168815 ± 181030 97 ± 85 28.13 ± 30.17

EXAMPLE 7 Testing the Effect of HGF Addition in Cardiomyocyte ProductionProcess

Originally HGF was included in the media as an “induction factor” priorto the transfer of the original plated-method from Research toDevelopment and was kept in as the protocol evolved from plated tosuspension aggregates. In the plated-method, previous studies had shownit was beneficial. Motivated by cost-reduction and processsimplification, the inventors revisited the need for HGF in the currentsuspension aggregate method. This Example summarizes a series ofexperiments that culminated in the optional role of HGF in the inductionmedia. This includes both the growth factor titration experiments thatled to the conclusion that HGF could be withdrawn without measurableimpact, which was confirmed by the qualification runs on a larger-scale.

Aggregate formation and cardiac induction procedures of those iPS cellsat different initial densities were performed essentially the same asdescribed in Examples 4 except different concentrations of human HGF(hHGF) and FGF were applied to Aggregate Formation Medium, AggregateTransition Medium and Cardiac Induction Medium. Experimental Design wassummarized in Tables 17-18.

TABLE 17 Summary of HGF experiments Experiment Experimental DesigniPS6.1 Growth Full matrix: hbFGF {0, 25 ng/mL} × Factor Titration #1 HGF{0, 5, 20, 50, 150 ng/mL} iPS6.1 Growth Full matrix: hbFGF {3, 6.25,12.5, 25, 50, 100 Factor Titration #2 ng/mL} × HGF {0, 20, 50 ng/mL}iPS6.1 “Four Full matrix: hbFGF {0, 25 ng/mL} × Corners” HGF {0, 50ng/mL} H9-TGZ Growth Full matrix: hbFGF {25, 50, 100, 150 ng/mL} ×Factor Titration #2 HGF {0, 20, 50} Note: Due to poor aggregateformation, triplicate flasks were consolidated on day 2and carried outin singlicate. H9-TGZ Growth Full matrix: hbFGF {25, 50, 100, 150 ng/mL}× Factor Titration #2 HGF {0, 20, 50} (repeat) iPS-MRB 1 L Parallel 1 Lspinner flasks with and without HGF spinner qualification #1 iPS-MRB 1 LParallel 1 L spinner flasks with and without HGF spinner qualification#2 iPS-MRB 1 L Parallel 1 L spinner flasks with and without HGF spinnerqualification #3

TABLE 18 Summary of experimental parameters of HGF experiments ParameterMethod Timepoint Cardiomyocyte purity Flow cytometry of cTNT, Day 14GFP, or RFP Cell yield CEDEX Day 14

This experiment clearly showed that FGF is required for the CMdifferentiation process to be successful. Conditions without FGF hadvirtually no surviving aggregates and no measurable cardiomyocytes(FIGS. 29A-B). On the other hand, all conditions with FGF yieldedcardiomyocytes independent of HGF concentration (FIGS. 29A-B). There wasno consistent trend in cardiomyocyte yield or iPS:CM ratio across therange of HGF concentrations tested.

Based on the results of the first experiment (see FIGS. 29A-B), thesecond iPS6.1 growth factor titration focused on the lower dose rangefor both FGF and HGF (FIGS. 30A-B). A very clear dose response wasobserved for FGF where too little FGF led to poor aggregate survival andhigh doses had survival but lacked cardiogenesis. Unlike the firstexperiment, a subtle trend was observed here in HGF concentration,particularly in the best performing FGF concentration conditions, whereproductivity increased with higher HGF concentrations. However, it isimportant to note that in no case did the omission of HGF lead to thefailure of a condition which was otherwise successful in the presence ofHGF.

Concurrent with the second iPS growth factor titration, a simpleexperiment (Table 19) was set up with iPS6.1 cells to test the 2×2matrix of 0 and 25 ng/mL FGF and 0 and 50 ng/mL HGF. The positiveconcentrations of FGF and HGF were chosen based on the current bestpractices for iPS6.1 cells at the time and were identical to theconditions used for cardiac induction of H1 cells in both the plated andsuspension processes. This experiment confirmed previous work showingthe necessity of FGF for the process. However, in this experiment,inclusion of HGF together with FGF reduced overall culture efficiencyversus the condition with FGF alone.

TABLE 19 Summary of “Four-corners” experiment. Each entry represents asingle T25 flask. Label Input count Harvest Count % cTNT+ # of CMs inputiPS:CM Million CMs/L iPS +/+ 5c 2420930 7.10E+04 6.3 4473 541 0.89 HGF +FGF {open oversize brace} iPS +/+ 5b 2420930 1.97E+05 11.7 23049 105 4.6iPS +/+ 5a 2420930 3.93E+05 12.5 49125 49 9.6 iPS +/− 6a 24209302.40E+04 1.5 360 6725 0.072 HGF only {open oversize brace} iPS +/− 6b2420930 0.00E+00 7 0 N/A 0 iPS +/− 6c 2420930 6.00E+03 0 0 N/A 0 iPS −/+7c 2420930 1.67E+06 11.2 187488 13 37 FGF only {open oversize brace} iPS−/+ 7b 2420930 1.71E+06 11.9 203371 12 41 iPS −/+ 7a 2420930 1.73E+0513.1 22663 107 4.5 iPS −/− 8a 2420930 1.80E+04 6.8 1224 1978 0.24 No GF{open oversize brace} iPS −/− 8b 2420930 0.00E+00 2.1 0 N/A 0 iPS −/− 8c2420930 0.00E+00 0 0 N/A 0

While the iPS6.1 optimization was underway, a separate series ofexperiments was conducted to optimize the growth factor concentrationsfor the H9-TGZ cells. Initial experiments had indicated that higherlevels of FGF were required for efficient cardiac induction of H9-TGZcells. Therefore, the FGF range tested was higher than that in therecent iPS6.1 experiments. This experiment confirmed that optimumcardiomyocyte production requires 100-150 ng/mL FGF with the H9-TGZcells (FIGS. 31A-C). No trend was observed in process efficiency acrossdifferent HGF concentrations.

Due to the issues with aggregate formation noted above, the H9-TGZgrowth factor titration experiment was repeated. In this repeat,exceptional cell survival and expansion led to overly dense cultures andlikely reduced final CM purity. However, it is important to note thatdespite the overall poor performance of the cultures, the usual andexpected decrease in performance at low FGF level was still discernable(FIGS. 32A-C). There was no observable trend of culture performanceacross the range of HGF concentrations tested and again, none of the “NoHGF” conditions with sufficient FGF failed to produce cardiomyocytes.

A meta-analysis across these experiments was performed to assess whetherthe inventors could detect any measurable benefit of including HGF inthe induction medium. This analysis reviewed all the availableexperiments for paired conditions with and without 50 ng/mL HGF.Conditions in which the control leg (with HGF) failed to produce greaterthan 1% cardiomyocytes were excluded. This left 14 paired conditions (11performed in triplicate within an experiment). To aid in this analysis,a process raw material cost model was employed to estimate the cost permillion cardiomyocytes under each condition. This model used the mostup-to-date raw material cost and process information available at thetime (˜Jun. 1, 2009). This analysis is summarized in Tables 20 and 21.The data suggest that on average, there is no measurable positive ornegative effect (i.e. effect greater than the experimental noise) of HGFin the inventors' cardiomyocyte production process.

TABLE 20 Summary of HGF vs no HGF comparisons. Total Head- FractionFraction with Fraction to-Head with better better iPS:CM with betterpairings CM/L @ 50 ratio @ $/CM @ 50 with purity ng/mL HGF 50 ng/mL HGFng/mL HGF Experiment >1% than 0 than 0 than 0 First iPS GF 4 3/4 3/4 3/4matrix Second iPS 4 2/4 2/4 0/4 GF matrix First iPS 4- 1 0/1 0/1 0/1corners H9-TGZ 4 2/4 2/4 1/4 Matrix #2 H9-TGZ 1 0/1 0/1 0/1 Matrix #2repeat Total 14  7/14  7/14  4/14

TABLE 21 Statistical analysis of HGF vs no HGF pairwise comparisons.Paired t-test values are shown for each performance metric (CM/L,iPS:CM, $/CM). In addition the best and worst case scenarios and theaverage and standard deviations are shown. CM/L iPS:CM $/CM HGF Conc(ng/mL) 0 50 0 50 0 50 p-value for paired t-test 0.28 0.28 0.20 Best 130140 9 7 77 90 Worst 8 1.5 135 712 918 9000 Average 41 49 45 100 340 1186StDev 37 50 43 189 292 2406

Three qualification runs were performed to confirm at large scale andwith iPS6.1-MRB cells that removal of HGF has no measurable effect. Theresults from these runs are summarized in Table 22. Of these runs, onlyRun #1 would be regarded as a normal run—the overall cell andcardiomyocyte yields were atypically low in Runs 2 and 3. In run 1, theyields were effectively the same independent of HGF condition. In runs 2and 3, higher, albeit still low, yields were obtained in the cultureswith HGF compared to those without. From this, the inventors concludedthat in a “normal” run, HGF addition has no effect but that insuboptimal cultures, HGF may improve culture outcomes.

TABLE 22 Qualification runs of HGF withdrawal Purity) CM/L iPS:CM (%RFP+ (millions) ratio Run +HGF −HGF +HGF −HGF +HGF −HGF #1 6.6 7.3 48.951.5 10:1 10:1 (Jun. 6, 2009) #2 3.7 1.3 17.5 3.6 29:1 140:1  (Jun. 10,2009) #3 2.6 2.2 10.0 5.1 50:1 98:1 (Jun. 14, 2009)

In summary, using iPS6.1 and H9-TGZ cells in extensive matrixexperiments, the inventors were unable to measure an effect of HGF oncardiomyocyte culture productivity. In qualification runs withiPS6.1-MRB cells at larger scale, the inventors saw some evidence thatHGF may partially rescue poor performing cultures but it didn't impactyield, purity, or efficiency in a normal, higher yielding culture. Basedon these results, HGF may be dispensable from the formulation for thecardiac induction medium.

EXAMPLE 8 Procedure for Optimizing Cardiomyocyte Differentiation fromDifferent Lines of Pluripotent Stem Cells and After Culture in DifferentStem Cell Maintenance Media

A high degree of variability has been documented when differentiatingdifferent pluripotent stem cell lines. This has been accepted in thefield as a “propensity” of PSC to form various tissues (such ascardiomyocytes, blood, hepatocytes or neurons). However, this propensitybetween lines is documented using the same differentiation procedure foreach cell line. The differentiation of PSCs to different lineagesrequires in vitro conditions that direct cells through developmentalstages and ultimately to a desired phenotype. A typical differentiationprocedure usually contains culture conditions that attempt to mimic thein vivo environment driving the development of a particular lineage,such as by the addition of specific growth factors. When differentiatedin vitro, a number of sources contribute to the growth factorenvironment, including: 1) endogenous expression from the cellsthemselves, 2) the serum or media that the pluripotent stem cells arecultured and/or subsequently differentiated in, and 3) the addition ofexogenous growth factors. In regard to the endogenous expression ofgrowth factors, this can arise from clone-to-clone variability and fromdifferences in the primary culture of the cells. All the sourcescontributing to the growth factor environment in a given differentiationculture must be accounted for and balanced in order to achieve anoptimal differentiation. This may require: 1) the addition ofantagonists to reduce the total signal in certain pathways, 2) theaddition of agonists to increase the total signal of certain pathways or3) combinations of agonists and or antagonists to optimize the signal.

This Example describes a procedure to maximize the differentiationpotential of multiple PSC lines or clones, grown in variable primaryculture conditions, to the cardiomyocyte lineage by manipulating salientsignaling pathways, including BMP and Activin/Nodal. This procedureincludes: 1) screening the cardiogenic potential of each cell line in anumber of conditions, followed by 2) customizing the differentiationprotocol for an individual PSC line.

The Manufacturing Procedure as described in the previous Examples isfollowed and modified as detailed below.

At any point during aggregate formation but preferably at day 3 ofdifferentiation, to the typical differentiation culture media (outlinedin the previous Manufacturing Procedure), various combinations of growthfactor agonists and antagonists were added. Including: a) No additionalgrowth factors; b) Variable concentrations of BMP4 alone. For example, 1ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml; c) Variable concentrations ofActivin alone. For example, 1 ng/ml, 3 ng/ml, 6 ng/ml, 12 ng/ml; d)Variable concentrations of Dorsomorphin; e) Variable concentrations ofSB-431542; f) Variable combinations of BMP4 and Activin.

Media were changed daily using the various conditions outlined in theprevious paragraph up to day 7 of differentiation .

After day 7 of differentiation, the standard Manufacturing Procedure asdescribed in the previous Examples was followed.

At days 6, 7 and 8 of differentiation, a small sample was harvested fromeach condition to monitor for expression of markers consistent withcardiac mesoderm by flow cytometry (Table 23). This information was usedto predict optimal culture conditions to differentiate cells into thecardiac lineages. Typically, a 30% KDR+/PDGFR-a+ population isconsistent with the successful induction of cardiac mesoderm.

At the endpoint of the assay, typically day 14 of differentiation, thecultures were harvested and analyzed for percent cardiomyocytes based onexpression of proteins consistent with cardiomyocyte development (Table23). In addition, a total cell count was determined.

For example, FIGS. 33A-D details the results of a growthfactor/inhibitor screen utilizing varying concentrations of BMP (1ng/mL, 10 ng/mL), dorsomorphin (2 uM, 0.2 uM), Activin A (1 ng/mL, 10ng/mL), SB-431542 (10 uM, 0.1 uM) and a combination of Activin A (6ng/mL) and BMP4 (10 ng/mL) (variable concentrations of compounds wereadded at days 3 through 7 of differentiation). Analysis of the cells atday 14 post aggregate formation (FIGS. 33C and 33D) shows a higherconcentration of Troponin T (CTNT) positive cells in the treatmentcomprising a combination of Activin A and BMP4. The presence of CTNT wasdetermined by flow cytometry using an anti-CTNT Antibody. As such, theresults of this experiment reveal that the optimized culture conditionsfor the stem cell clone iPS 6.1 MRB for differentiation intocardiomyocytes is a combination of Activin A (6 ng/mL) and BMP4 (10ng/mL).

Based on marker analysis (for example, using flow cytometry) from days6, 7, 8, 9, 10 and/or at earlier or later time points in differentiationand optionally the total yield of cardiomyocytes, the cultures that hadthe highest yield and/or purity (or another measure of optimal cellculture growth and/or differentiation for the desired differentiatedcell type, such as function of a cell-specific enzyme or receptor orelectrophysiological function specific for the desired cell type) wereidentified and, therefore, the corresponding culture conditions areknown that resulted in optimal differentiation of the pluripotent stemcell clone utilized. These culture conditions can then be routinelyutilized during the Manufacturing Procedure, thereby coupling thepluripotent stem cells from the same cell line or clone with the sameculture medium composition, or further manipulations can be investigatedto increase the yield.

TABLE 23 Non-limiting Examples of Growth Factors and Markers associatedwith developmental stages of Cardiac Mesoderm and CardiomyocytesExamples Examples of Growth of Cardiac Examples of Growth Examples ofFactors that induce Mesoderm Factors specifying Cardiomyocyte CardiacMesoderm Markers cardiomyocytes Markers Wnt KDR BMP2 NKX2.5ActivinA/Nodal PDGFR-a BMP4 TBX5 BMP2 CXCR4 BMP10 GATA4 BMP4 CKITnegActivinA/Nodal Baf60c BMP10 N-Cadherin bFGF alpha-MHC bFGF MESP1 EGFCTNT IGF IGF MLC2A (Wnt inhibitors) MLC2V MLC1V Sarcomeric alpha-actininNPPA Abbreviations and definition PSC = Pluripotent Stem Cells -Embryonic and Induced Pluripotent Stem Cells. BMP4 = Bone MorphogenicProtein-4; Developmental morphogen Activin = ActivinA (Activin and Nodalsignaling can be used interchangeably in this context). Developmentalmorphogen SB-431542 = Small molecule inhibitor of the TGFβ/Activin/Nodalsignaling pathway Dor = Dorsomorphin. Small molecule inhibitor of theBMP pathway. Also known as Compound C KDR = Kinase Insert DomainReceptor, also known as VEGFR-2 PDGFR-a = Platelet Derived Growth FactorReceptor-alpha

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for increased production ofcardiomyocytes, wherein the method comprises the steps of: a) incubatingpluripotent stem cells from a selected cell clone in a suspensionculture under conditions to promote aggregate formation; and b)differentiating the stem cells into cardiomyocytes in a cardiacdifferentiation medium prepared from a selected batch of culture medium,the differentiation medium having been prepared from said culture mediumby adjusting the level of one or more differentiation factors in saidculture medium at amounts determined to be appropriate for cardiacdifferentiation of the selected cell clone and culture media batchemployed.
 2. The method of claim 1, wherein the selected cell clone isan induced pluripotent stem (iPS) cell clone.
 3. The method of claim 1,wherein the selected cell clone is derived from a single pluripotentstem cell in an adherent culture.
 4. The method of claim 3, wherein theselected cell clone is derived from a single pluripotent stem cell by aprocess comprising incubating the single pluripotent stem cell in mediumcomprising a Rho-associated kinase (ROCK) inhibitor or a myosin IIinhibitor under conditions to promote cell growth.
 5. The method ofclaim 1, wherein from about 10⁶ to about 10¹⁰ of the pluripotent stemcells are first incubated in the suspension culture in step a).
 6. Themethod of claim 1, wherein the conditions to promote aggregate formationcomprises externally added ROCK inhibitor, myosin II inhibitor,fibroblast growth factor (FGF) or hepatic growth factor (HGF).
 7. Themethod of either of claim 4 or 6, wherein the myosin II inhibitor isblebbistatin.
 8. The method of claim 1, wherein the aggregates formedfrom the pluripotent stem cells prior to cardiac differentiation areabout 10 nm to 400 μm in diameter.
 9. The method of claim 1, wherein thesuspension culture for differentiating the stem cells has a volume offrom 5 milliliters to 25 liters.
 10. The method of claim 1, wherein thepluripotent stem cells and/or cardiomyocytes differentiated therefromcontain one or more transgenes.
 11. The method of claim 10, wherein theone or more transgenes encode a selectable and/or screenable markerunder the control of a cardiomyocyte-specific promoter.
 12. The methodof claim 1, further comprising enriching or purifying the differentiatedcardiomyocytes.
 13. The method of claim 1, wherein the cardiacdifferentiation medium comprises externally added fibroblast growthfactor (FGF) in an amount of from 5 to 200 ng/ml.
 14. The method ofclaim 1, wherein the suspension culture is rotated or shaken at a speedof about 15 rpm to 100 rpm.
 15. The method of claim 1, wherein theculture medium is TeSR, mTeSR, RPMI medium, supplemented DMEM-F12 ordilutions thereof.
 16. The method of claim 1, wherein thedifferentiation factors whose level are adjusted in the culture mediumcomprise one or more of modulators of signaling pathways of bonemorphogenetic protein, ActivinA/Nodal, vascular endothelial growthfactor (VEGF), dickkopf homolog 1 (DKK1), basic fibroblast growth factor(bFGF), insulin growth factor (IGF), and/or epidermal growth factor(EGF).
 17. The method of claim 16, wherein the differentiation factorscomprises BMP2, BMP4, BMP10, Activin A, bFGF, IGF, EGF, BMP signalinginhibitor, Activin signaling inhibitor, or a combination thereof. 18.The method of claim 17, wherein the BMP signaling inhibitor comprisesdorsomorphin.
 19. The method of claim 17, wherein the Activin Asignaling inhibitor comprises SB431542.
 20. An isolated cell populationof about 5×10⁸ to 10¹⁰ cells comprising at least 99% cardiomyocytes.